gene therapy essay conclusion

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CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future

Fathema uddin.

1 Department of Medicine, Thoracic Oncology Service, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Charles M. Rudin

2 Weill Cornell Medicine, Cornell University, New York, NY, United States

Triparna Sen

A series of recent discoveries harnessing the adaptive immune system of prokaryotes to perform targeted genome editing is having a transformative influence across the biological sciences. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is redefining our approach to gene therapy. Traditional gene therapy has raised some concerns, as its reliance on viral vector delivery of therapeutic transgenes can cause both insertional oncogenesis and immunogenic toxicity. While viral vectors remain a key delivery vehicle, CRISPR technology provides a relatively simple and efficient alternative for site-specific gene editing, obliviating some concerns raised by traditional gene therapy. Although it has apparent advantages, CRISPR/Cas9 brings its own set of limitations which must be addressed for safe and efficient clinical translation. This review focuses on the evolution of gene therapy and the role of CRISPR in shifting the gene therapy paradigm. We review the emerging data of recent gene therapy trials and consider the best strategy to move forward with this powerful but still relatively new technology.

Introduction

Gene therapy as a strategy to provide therapeutic benefit includes modifying genes via disruption, correction, or replacement ( 1 ). Gene therapy has witnessed both early successes and tragic failures in a clinical setting. The discovery and development of the CRISPR/Cas9 system has provided a second opportunity for gene therapy to recover from its stigma and prove to be valuable therapeutic strategy. The recent advent of CRISPR technology in clinical trials has paved way for the new era of CRISPR gene therapy to emerge. However, there are several technical and ethical considerations that need addressing when considering its use for patient care. This review aims to (1) provide a brief history of gene therapy prior to CRISPR and discuss its ethical dilemmas, (2) describe the mechanisms by which CRISPR/Cas9 induces gene edits, (3) discuss the current limitations and advancements made for CRISPR technology for therapeutic translation, and (4) highlight a few recent clinical trials utilizing CRISPR gene therapy while opening a discussion for the ethical barriers that these and future trials may hinge upon.

Gene Therapy Prior to Crispr—History, Hurdles, and its Future

Origins of gene therapy.

The introduction of gene therapy into the clinic provided hope for thousands of patients with genetic diseases and limited treatment options. Initially, gene therapy utilized viral vector delivery of therapeutic transgenes for cancer treatment ( 2 ) or monogenic disease ( 3 ). One of these pioneering clinical trials involved ex vivo retroviral delivery of a selective neomycin-resistance marker to tumor infiltrating leukocytes (TILs) extracted from advanced melanoma patients ( 4 ). Although the neomycin tagging of TILs did not have a direct therapeutic intent and was used for tracking purposes, this study was the first to provide evidence for both the feasibility and safety of viral-mediated gene therapy. Soon after, the first clinical trial that used gene therapy for therapeutic intent was approved in 1990 for the monogenic disease adenosine deaminase-severe combined immunodeficiency (ADA-SCID). Two young girls with ADA-SCID were treated with retroviruses for ex vivo delivery of a wildtype adenosine deaminase gene to autologous T-lymphocytes, which were then infused back into the patients ( 5 , 6 ). While one patient showed moderate improvement, the other did not ( 5 , 6 ) Although initial results were suboptimal, the early evidence of feasibility prompted multiple subsequent gene therapy trials using viral-mediated gene edition. However, this was followed by some major setbacks.

Tragic Setbacks for Gene Therapy

Jesse Gelsinger, an 18-year-old with a mild form of the genetic disease ornithine transcarbamylase (OTC) deficiency, participated in a clinical trial which delivered a non-mutated OTC gene to the liver through a hepatic artery injection of the recombinant adenoviral vector housing the therapeutic gene. Unfortunately, Jesse passed away 4 days after treatment ( 7 ). The adenovirus vector triggered a much stronger immune response in Jesse than it had in other patients, causing a chain of multiple organ failures that ultimately led to his death ( 8 ). At the time of the trial, adenoviral vectors were considered reasonably safe. In preclinical development, however, two of the rhesus monkeys treated with the therapy developed a similar pattern of fatal hepatocellular necrosis ( 9 ). Shortly after, another gene therapy trial led to the development of leukemia in several young children induced by insertional oncogenesis from the therapy ( 10 ). These trials opened for two forms of SCID (SCID-X1 or common ɤ chain deficiency) and adenosine deaminase deficiency (ADA). The therapy used ɤ-retroviral vectors for ex vivo delivery of therapeutic transgenes to autologous CD34+ hematopoietic stem cells, which were reintroduced to the patients ( 10 ). Five patients developed secondary therapy-related leukemia, one of whom died from the disease ( 11 ). Further investigation revealed integration of the therapeutic gene into the LMO2 proto-oncogene locus, presumably resulting in the development of leukemia ( 12 ). Subsequent analyses have suggested a higher frequency of insertional mutagenesis events with ɤ-retroviral vectors relative to other vectors ( 13 ). Together, these tragic events prompted substantial post-hoc concerns regarding the nature of appropriate informed consent and the stringency of safety and eligibility parameters for gene therapy experimentation in humans ( 14 ).

Shifting the Gene Therapy Paradigm

Almost two decades after these cases, gene therapy returned in clinical trials with reengineered viruses designed with safety in mind. Current clinical approaches are being scrutinized for evidence of insertional mutagenesis and adverse immunogenic reactions ( 15 – 18 ). Non-viral vectors have been used as an alternative method for gene delivery, which have reduced immunogenicity compared to their viral counterparts and therefore greater tolerance for repeated administration. A concern is whether these methods can be optimized to provide equivalent efficiency of gene delivery to that provided by viruses ( 19 ).

While viral vectors continue to be essential for current gene therapy, the concerns and limitations of viral-mediated gene edition has broadened the diversity of gene-editing approaches being considered. Rather than introducing the therapeutic gene into a novel (and potentially problematic) locus, a more attractive strategy would be to directly correct the existing genetic aberrations in situ . This alternative would allow the pathological mutation to be repaired while averting the risk of insertional oncogenesis. The discovery and repurposing of nucleases for programmable gene editing made this possible, beginning with the development of zinc finger nucleases (ZFN) ( 20 , 21 ), followed by transcription activator-like effector nucleases (TALENs), meganucleases, and most recently, the CRISPR/Cas system ( 22 ). While the other gene-editing tools can induce genome editing at targeted sites under controlled conditions, the CRISPR/Cas system has largely supplanted these earlier advances due to its relatively low price, ease of use, and efficient and precise performance. However, this technology is often delivered with adeno-associated virus (AAV) vectors, and thus does not completely avert risks associated with viruses. Other delivery options are available to circumvent this issue, each with their own advantages and challenges (see Delivery of CRISPR Gene Therapy section). Of the CRISPR/Cas systems, CRISPR/Cas9 is the most developed and widely used tool for current genome editing.

CRISPR/Cas9 Mediated Gene Editing

Pioneering discoveries in crispr/cas9 technology.

The bacterial CRISPR locus was first described by Francisco Mojica ( 23 ) and later identified as a key element in the adaptive immune system in prokaryotes ( 24 ). The locus consists of snippets of viral or plasmid DNA that previously infected the microbe (later termed “spacers”), which were found between an array of short palindromic repeat sequences. Later, Alexander Bolotin discovered the Cas9 protein in Streptococcus thermophilus , which unlike other known Cas genes, Cas9 was a large gene that encoded for a single-effector protein with nuclease activity ( 25 ). They further noted a common sequence in the target DNA adjacent to the spacer, later known as the protospacer adjacent motif (PAM)—the sequence needed for Cas9 to recognize and bind its target DNA ( 25 ). Later studies reported that spacers were transcribed to CRISPR RNAs (crRNAs) that guide the Cas proteins to the target site of DNA ( 26 ). Following studies discovered the trans-activating CRISPR RNA (tracrRNA), which forms a duplex with crRNA that together guide Cas9 to its target DNA ( 27 ). The potential use of this system was simplified by introducing a synthetic combined crRNA and tracrRNA construct called a single-guide RNA (sgRNA) ( 28 ). This was followed by studies demonstrating successful genome editing by CRISPR/Cas9 in mammalian cells, thereby opening the possibility of implementing CRISPR/Cas9 in gene therapy ( 29 ) ( Figure 1 ).

Hallmarks of CRISPR Gene Therapy. Timeline highlighting major events of traditional gene therapy, CRISPR development, and CRISPR gene therapy. The text in red denotes gene therapy events which have raised significant ethical concerns.

Mechanistic Overview of CRISPR/Cas9-Mediated Genome Editing

CRISPR/Cas9 is a simple two-component system used for effective targeted gene editing. The first component is the single-effector Cas9 protein, which contains the endonuclease domains RuvC and HNH. RuvC cleaves the DNA strand non-complementary to the spacer sequence and HNH cleaves the complementary strand. Together, these domains generate double-stranded breaks (DSBs) in the target DNA. The second component of effective targeted gene editing is a single guide RNA (sgRNA) carrying a scaffold sequence which enables its anchoring to Cas9 and a 20 base pair spacer sequence complementary to the target gene and adjacent to the PAM sequence. This sgRNA guides the CRISPR/Cas9 complex to its intended genomic location. The editing system then relies on either of two endogenous DNA repair pathways: non-homologous end-joining (NHEJ) or homology-directed repair (HDR) ( Figure 2 ). NHEJ occurs much more frequently in most cell types and involves random insertion and deletion of base pairs, or indels, at the cut site. This error-prone mechanism usually results in frameshift mutations, often creating a premature stop codon and/or a non-functional polypeptide. This pathway has been particularly useful in genetic knock-out experiments and functional genomic CRISPR screens, but it can also be useful in the clinic in the context where gene disruption provides a therapeutic opportunity. The other pathway, which is especially appealing to exploit for clinical purposes, is the error-free HDR pathway. This pathway involves using the homologous region of the unedited DNA strand as a template to correct the damaged DNA, resulting in error-free repair. Experimentally, this pathway can be exploited by providing an exogenous donor template with the CRISPR/Cas9 machinery to facilitate the desired edit into the genome ( 30 ).

CRISPR/Cas9 mediated gene editing. Cas9 in complex with the sgRNA targets the respective gene and creates DSBs near the PAM region. DNA damage repair proceeds either through the NHEJ pathway or HDR. In the NHEJ pathway, random insertions and deletions (indels) are introduced at the cut side and ligated resulting in error-prone repair. In the HDR pathway, the homologous chromosomal DNA serves as a template for the damaged DNA during repair, resulting in error-free repair.

Limitations and Advancements of CRISPR/Cas9

Off-target effects.

A major concern for implementing CRISPR/Cas9 for gene therapy is the relatively high frequency of off-target effects (OTEs), which have been observed at a frequency of ≥50% ( 31 ). Current attempts at addressing this concern include engineered Cas9 variants that exhibit reduced OTE and optimizing guide designs. One strategy that minimizes OTEs utilizes Cas9 nickase (Cas9n), a variant that induces single-stranded breaks (SSBs), in combination with an sgRNA pair targeting both strands of the DNA at the intended location to produce the DSB ( 32 ). Researchers have also developed Cas9 variants that are specifically engineered to reduce OTEs while maintaining editing efficacy ( Table 1 ). SpCas9-HF1 is one of these high-fidelity variants that exploits the “excess-energy” model which proposes that there is an excess affinity between Cas9 and target DNA which may be enabling OTEs. By introducing mutations to 4 residues involved in direct hydrogen bonding between Cas9 and the phosphate backbone of the target DNA, SpCas9-HF1 has been shown to possess no detectable off-target activity in comparison to wildtype SpCas9 ( 35 ). Other Cas9 variants that have been developed include evoCas9 and HiFiCas9, both of which contain altered amino acid residues in the Rec3 domain which is involved in nucleotide recognition. Desensitizing the Rec3 domain increases the dependence on specificity for the DNA:RNA heteroduplex to induce DSBs, thereby reducing OTEs while maintaining editing efficacy ( 38 , 39 ). One of the more recent developments is the Cas9_R63A/Q768A variant, in which the R63A mutation destabilizes R-loop formation in the presence of mismatches and Q768A mutation increases sensitivity to PAM-distal mismatches ( 49 ). Despite the different strategies, the rational for generating many Cas9 variants with reduced OTEs has been to ultimately reduce general Cas9 and DNA interactions and give a stronger role for the DNA:RNA heteroduplex in facilitating the edits.

Cas9 variants.

Optimizing guide designs can also reduce the frequency of OTEs ( 31 ). Many features in an sgRNA determine specificity including the seed sequence (a 10–12 bp region proximal to PAM on 3′ of spacer sequence) ( 29 , 53 ), GC content ( 54 , 55 ), and modifications such as 5′ truncation of the sgRNA ( 56 ). Several platforms have also been designed to provide optimized guide sequences against target genes, including E-Crisp ( 31 , 57 ), CRISPR-design, CasOFFinder, and others ( 31 ). However, many of these tools are designed based on computational algorithms with varying parameters or rely on phenotypic screens that may be specific to cell types and genomes, generating appreciable noise and lack of generalizability across different experimental setups ( 58 , 59 ). Recently, an additional guide design tool named sgDesigner was developed that addressed these limitations by employing a novel plasmid library in silico that contained both the sgRNA and the target site within the same construct. This allowed collecting Cas9 editing efficiency data in an intrinsic manner and establish a new training dataset that avoids the biases introduced through other models. Furthermore, a comparative performance evaluation to predict sgRNA efficiency of sgDesigner with 3 other commonly used tools (Doench Rule Set 2, Sequence Scan for CRISPR and DeepCRISPR) revealed that sgDesigner outperformed all 3 designer tools in 6 independent datasets, suggesting that sgDesigner may be a more robust and generalizable platform ( 60 ).

Protospacer Adjacent Motif Requirement

An additional limitation of the technology is the requirement for a PAM near the target site. Cas9 from the bacteria Streptococcus pyogenes (SpCas9) is one of the most extensively used Cas9s with a relatively short canonical PAM recognition site: 5′NGG3′, where N is any nucleotide. However, SpCas9 is relatively large and difficult to package into AAV vectors ( 61 , 62 ), the most common delivery vehicle for gene therapy. Staphylococcus aureus Cas9 (SaCas9) is a smaller ortholog that can be packaged more easily in AAV vectors but has a longer PAM sequence: 5′NNGRRT3′ or 5′NNGRR(N)3′, where R is any purine, which further narrows the window of therapeutic targeting sites. Engineered SaCas9 variants have been made, such as KKH SaCas9, which recognizes a 5′NNNRRT3′ PAM, broadening the human targeting sites by 2- to 4-fold. OTEs, however, are observed with frequencies similar to wildtype SaCas9 and need to be considered in designing any therapeutic application ( 33 ). Several other variants of SpCas9 have also been engineered for broadening the gene target window including SpCas9-NG, which recognizes a minimal NG PAM ( 44 ) and xCas9, which recognizes a broad range of PAM including NG, GAA, and GAT ( 43 ). A side by side comparison of both variants revealed that while SpCas9-NG had a broader PAM recognition, xCas9 had the lowest OTE in human cells ( 63 ). Another Cas9 ortholog from the bacteria Streptococcus canis , ScCas9, has been recently characterized with a minimal PAM specificity of 5′NNG3′ and an 89.2% sequence homology to SpCas9 and comparable editing efficiency to SpCas9 in both bacterial and human cells ( 52 ). The most recent development is a variant of SpCas9 named SpRY that has been engineered to be nearly PAMless, recognizing minimal NRN > NYN PAMs. This new variant can potentially edit any gene independent of a PAM requirement, and hence can be used therapeutically against several genetic diseases ( 47 ).

Alternatively, RNA-targeting Cas9 variants have been developed which also broaden the gene targeting spectrum by mitigating PAM requirement restrictions. S. pyogenese Cas9 (SpyCas9) can be manipulated to target RNA by providing a short oligonucleotide with a PAM sequence, known as a PAMmer ( 64 , 65 ), and thus eliminates the need for a PAM site within the target region. Other subsets of Cas enzymes have also been discovered that naturally target RNA independent of a PAM, such as Cas13d. Upon further engineering of this effector, CasRx was developed for efficient RNA-guided RNA targeting in human cells ( 66 , 67 ). Although RNA-targeting CRISPR advances provide a therapeutic opportunity without the risk of DNA-damage toxicity, they exclude the potential for editing a permanent correction into the genome.

DNA-Damage Toxicity

CRISPR-induced DSBs often trigger apoptosis rather than the intended gene edit ( 68 ). Further safety concerns were revealed when using this tool in human pluripotent stem cells (hPSCs) which demonstrated that p53 activation in response to the toxic DSBs introduced by CRISPR often triggers subsequent apoptosis ( 69 ). Thus, successful CRISPR edits are more likely to occur in p53 suppressed cells, resulting in a bias toward selection for oncogenic cell survival ( 70 ). In addition, large deletions spanning kilobases and complex rearrangements as unintended consequences of on-target activity have been reported in several instances ( 71 , 72 ), highlighting a major safety issue for clinical applications of DSB-inducing CRISPR therapy. Other variations of Cas9, such as catalytically inactive endonuclease dead Cas9 (dCas9) in which the nuclease domains are deactivated, may provide therapeutic utility while mitigating the risks of DSBs ( 73 ). dCas9 can transiently manipulate expression of specific genes without introducing DSBs through fusion of transcriptional activating or repressing domains or proteins to the DNA-binding effector ( 74 ). Other variants such as Cas9n can also be considered, which induces SSBs rather than DSBs. Further modifications of these Cas9 variants has led to the development of base editors and prime editors, a key innovation for safe therapeutic application of CRISPR technology (see Precision Gene Editing With CRISPR section).

Immunotoxicity

In addition to technical limitations, CRISPR/Cas9, like traditional gene therapy, still raises concerns for immunogenic toxicity. Charlesworth et al. showed that more than half of the human subjects in their study possessed preexisting anti-Cas9 antibodies against the most commonly used bacterial orthologs, SaCas9 and SpCas9 ( 75 ). Furthermore, AAV vectors are also widely used to deliver CRISPR components for gene therapy. To this end, several Cas9 orthologs and AAV serotypes were tested based on sequence similarities and predicted binding strength to MHC class I and class II to screen for immune orthologs that can be used for safe repeated administration of AAV-CRISPR gene therapy. Although no two AAV serotypes were found to completely circumvent immune recognition, the study verified 3 Cas9 orthologs [SpCas9, SaCas9, and Campylobacter jejuni Cas9 (CjCas9)] which showed robust editing efficiency and tolerated repeated administration due to reduced immunogenic toxicity in mice immunized against AAV and Cas9 ( 76 ). A major caveat is pre-existing immunity in humans against 2 of these orthologs—SpCas9 and SaCas9, leaving CjCas9 as the only current option for this cohort of patients. However, this ortholog has not been well-studied in comparison to the other 2 orthologs and will need further investigation to provide evidence for its safety and efficacy for clinical use. Future studies may also identify other Cas9 immune-orthogonal orthologs for safe repeated gene therapy.

Precision Gene Editing With CRISPR

Precise-genome editing is essential for prospects of CRISPR gene therapy. Although HDR pathways can facilitate a desired edit, its low efficiency renders its utility for precise gene editing for clinical intervention highly limiting, with NHEJ as the default pathway human cells take for repair. Enhancement of HDR efficiency has been achieved via suppression of the NHEJ pathway through chemical inhibition of key NHEJ modulating enzymes such as Ku ( 77 ), DNA ligase IV ( 78 ), and DNA-dependent protein kinases (DNA-PKcs) ( 79 ). Other strategies that improve HDR efficiency include using single-stranded oligodeoxynucleotide (ssODN) template, which contains the homology arms to facilitate recombination and the desired edit sequence, instead of double-stranded DNA (dsDNA). Rationally designed ssODN templates with optimized length complementarity have been shown to increase HDR rates up to 60% in human cells for single nucleotide substitution ( 80 ). Furthermore, cell cycle stage plays a key role in determining the DNA-damage repair pathway a cell may take. HDR events are generally restricted to late S and G2 phases of the cell cycle, given the availability of the sister chromatid to serve as a template at these stages, whereas NHEJ predominates the G1, S, and G2 phases ( 81 ). Pharmacological arrest at the S phase with aphidicolin increased HDR frequency in HEK293T with Cas9-guide ribonucleoprotein (RNP) delivery. Interestingly, cell arrest in the M phase using nocodazole with low concentrations of the Cas9-guide RNP complex yielded higher frequencies of HDR events in these cells, reaching a maximum frequency of up to 31% ( 82 ). Although HDR is considered to be restricted to mitotic cells, a recent study revealed that the CRISPR/Cas9 editing can achieve HDR in mature postmitotic neurons. Nishiyama et al. successfully edited the CaMKIIα locus through HDR in postmitotic hippocampal neurons of adult mice in vitro using an AAV delivered Cas9, guide RNA, and donor template in the CaMKIIα locus, which achieved successful HDR-mediated edits in ~30% of infected cells. Although HDR efficiency was dose-dependent on AAV delivered HDR machinery and off-target activity was not monitored, this study demonstrated CRISPR's potential utility for translational neuroscience after further developments ( 83 ). To further exploit cell-cycle stage control as a means to favor templated repair, Cas9 conjugation to a part of Geminin, a substrate for G1 proteosome degradation, can limit Cas9 expression to S, G2, and M stages. This strategy was shown to facilitate HDR events while mitigating undesired NHEJ edits in human immortalized and stem cells ( 84 , 85 ). A more recent strategy combined a chemically modified Cas9 to the ssODN donor or a DNA adaptor that recruits the donor template, either of which improved HDR efficiency by localizing the donor template near the cleavage site ( 86 ). Despite these advancements, HDR is still achieved at a relatively low efficiency in eukaryotic cells and use of relatively harmful agents in cells such as NHEJ chemical inhibitors may not be ideal in a clinical setting.

A recent advancement that allows precision gene editing independent of exploiting DNA damage response mechanisms is the CRISPR base editing (BE) system. In this system, a catalytically inactive dead Cas9 (dCas9) is conjugated to deaminase, which can catalyze the conversion of nucleotides via deamination. For increased editing efficiency, Cas9 nickase (Cas9n) fused with deaminase is recently being more utilized over dCas9 for base editing, as the nicks created in a single strand of DNA induce higher editing efficiency. Currently, the two types of CRISPR base editors are cytidine base editors (CBEs) and adenosine base editors (ABEs). CBEs catalyze the conversion of cytidine to uridine, which becomes thymine after DNA replication. ABEs catalyze the conversion of adenosine to inosine which becomes guanine after DNA replication ( 87 ). Base editors provide a means to edit single nucleotides without running the risk of causing DSB-induced toxicity. However, base editors are limited to “A to T” and “C to G” conversions, narrowing its scope for single-base gene edition to only these bases. In addition, base editors still face some of the same challenges as the previously described CRISPR systems, including OTEs, more so with CBEs than ABEs ( 88 , 89 ) and packaging constraints, namely in AAV vectors due to the large size of base editors ( 90 ). Furthermore, the editing window for base editors are limited to a narrow range of a few bases upstream of the PAM ( 90 ). More recently, prime editing has been developed as a strategy to edit the genome to insert a desired stretch of edits without inducing DSBs ( 91 ). This technology combines fusion of Cas9n with a reverse transcriptase and a prime editing guide RNA (pegRNA), which contains sgRNA sequence, primer binding site (PBS), and an RNA template encoding the desired edit on the 3′ end. Prime editors use Cas9n to nick one strand of the DNA and insert the desired edit via reverse transcription of the RNA template. The synthesized edit is incorporated into the genome and the unedited strand is cleaved and repaired to match the inserted edit. With an optimized delivery system in place, base editors and primer editors can open the door for precision gene editing to correct and potentially cure a multitude of genetic diseases ( Figure 3 ).

Precise Gene Editing. (A) CRISPR/Cas9-HDR. Cas9 induces a DSB. The exogenous ssODN carrying the sequence for the desired edit and homology arms is used as a template for HDR-mediated gene modification. (B) Base Editor. dCas9 or Cas9n is tethered to the catalytic portion of a deaminase. Cytosine deaminase catalyzes the formation of uridine from cytosine. DNA mismatch repair mechanisms or DNA replication yield an C:G to T:A single nucleotide base edit. Adenosine deaminase catalyzes the formation of inosine from adenosine. DNA mismatch repair mechanisms or DNA replication yield an A:T to G:C single nucleotide base edit. (C) Prime Editor. Cas9n is tethered to the catalytic portion of reverse transcriptase. The prime editor system uses pegRNA, which contains the guide spacer sequence, reverse transcriptase primer, which includes the sequence for the desired edit and a primer binding site (PBS). PBS hybridizes with the complementary region of the DNA and reverse transcriptase transcribes new DNA carrying the desired edit. After cleavage of the resultant 5′ flap and ligation, DNA repair mechanisms correct the unedited strand to match the edited strand. HDR, homology directed repair. DSB, double stranded break; SSB, single-stranded break; ssODN, single-stranded oligodeoxynucleotide.

Delivery of CRISPR Gene Therapy

The delivery modality of CRISPR tools greatly influences its safety and therapeutic efficacy. While traditional gene therapy utilizing viruses have been scrutinized for the risk of immunotoxicity and insertional oncogenesis, AAV vectors remain a key delivery vehicle for CRISPR gene therapy and continues to be extensively used for its high efficiency of delivery ( 92 ). The CRISPR toolkit can be packaged as plasmid DNA encoding its components, including Cas9 and gRNA, or can be delivered as mRNA of Cas9 and gRNA. Nucleic acids of CRISPR can be packaged in AAV vectors for delivery or introduced to target cells via electroporation/nucleofection or microinjection, with the latter methods averting virus-associated risks. However, microinjection can be technically challenging and is only suited for ex vivo delivery. Electroporation is also largely used for ex vivo but can be used in vivo for certain target tissues ( 93 ). However, high-voltage shock needed to permeabilize cell membranes via electroporation can be toxic and can lead to permanent permeabilization of treated cells ( 94 ). In addition to viral toxicity, AAV delivery of CRISPR components yields longevity of expression, leading to greater incidence of OTEs. Alternatively, delivery of the Cas9 protein and gRNA as RNP complexes has reduced OTEs while maintained editing efficacy, owing to its transient expression and rapid clearance in the cell ( 95 ).

Once the delivery modality is selected, CRISPR/Cas9 edits can be facilitated either ex vivo where cells are genetically modified outside of the patient and reintroduced back, or in vivo with delivery of the CRISPR components directly into the patient where cells are edited ( Figure 4 ). Both systems pose their own set of advantages and challenges. Advantages for ex vivo delivery include greater safety since patients are not exposed to the gene altering tool, technical feasibility, and tighter quality control of the edited cells. However, challenges to this method include survival and retention of in vivo function of cells outside the patient after genetic manipulation and extensive culture in vitro . Also, an adequate supply of cells is needed for efficient re-engraftment. These conditions limit this method to certain cell types that can survive and be expanded in culture, such as hematopoietic stem and progenitor cells (HSPCs) ( 96 ) and T cells ( 97 ).

Delivery of CRISPR Therapy. Nucleic acids encoding CRISPR/Cas9 or its RNP complex can be packaged into delivery vehicles. Once packaged, edits can be facilitated either ex vivo or in vivo . Ex vivo editing involves extraction of target cells from the patient, cell culture, and expansion in vitro , delivery of the CRISPR components to yield the desired edits, selection, and expansion of edited cells, and finally reintroduction of therapeutic edited cells into the patient. In vivo editing can be systemically delivered via intravenous infusions to the patient, where the CRISPR cargo travels through the bloodstream via arteries leading to the target tissue, or locally delivered with injections directly to target tissue. Once delivered, the edits are facilitated in vivo to provide therapeutic benefit.

While ex vivo gene therapy has provided therapeutic benefit for hematological disorders and cancer immunotherapy, many tissue types are not suited for this method, severely limiting its therapeutic utility for other genetic diseases. in vivo manipulation is thus needed to expand CRISPR's utility to treat a broader range of genetic diseases, such as Duchenne muscular dystrophy (DMD) ( 98 ) and hereditary tyrosinemia ( 99 ). CRISPR components can be delivered in vivo systemically through intravenous injections or can be locally injected to specific tissues ( Figure 4 ). With systemic delivery, the CRISPR components and its vehicle are introduced into the circulatory system where expression of the gene editing toolkit can be controlled to target specific organs via tissue-specific promoters ( 100 ). However, challenges of in vivo delivery include degradation by circulating proteases or nucleases, opsonization by opsonins, or clearance by the mononuclear phagocyte system (MPS). Furthermore, the cargo must reach the target tissue and bypass the vascular endothelium, which are often tightly connected by cell-cell junctions ( 101 ), preventing accessibility to larger delivery vehicles (>1 nm diameter). Additionally, once the cargo has reached the target cells, they must be internalized, which is generally facilitated through endocytosis where they can be transported and degraded by lysosomal enzymes ( 102 ). In addition, localization of the editing machinery near the point of injection can result in uneven distribution of the edited cell repertoire within the tissue, which may result in suboptimal therapeutic outcomes ( 102 ). While advancements are continuing to refine delivery techniques, the current systems have allowed CRISPR gene therapy to be used in the clinic.

Biological Intervention of CRISPR/Cas9 in Clinical Trials

Cancer immunotherapy.

The first CRISPR Phase 1 clinical trial in the US opened in 2018 with the intent to use CRISPR/Cas9 to edit autologous T cells for cancer immunotherapy against several cancers with relapsed tumors and no further curative treatment options. These include multiple myeloma, melanoma, synovial sarcoma and myxoid/round cell liposarcoma. This trial was approved by the United States Food and Drug Administration (FDA) after careful consideration of the risk to benefit ratios of this first application of CRISPR gene therapy into the clinic. During this trial, T lymphocytes were collected from the patients' blood and ex vivo engineered with CRISPR/Cas9 to knockout the α and β chains of the endogenous T cell receptor (TCR), which recognizes a specific antigen to mediate an immune response, and the programmed cell death-1 (PD-1) protein, which attenuates immune response. The cells were then transduced with lentivirus to deliver a gene encoding a TCR specific for a NY-ESO-1 antigen, which has been shown to be highly upregulated in the relapsed tumors and thus can serve as a therapeutic target. Since then, many trials have opened for CRISPR-mediated cancer immunotherapy and is currently the most employed strategy for CRISPR gene therapy ( Table 2 ). A trial implementing this strategy using other tools had already been conducted in both pre-clinical and clinical settings, but this was the first time CRISPR/Cas9 was used to generate the genetically modified T cells ( 97 ). The moderate transition of switching only the tool used for an already approved therapeutic strategy may have been key to paving the road for using CRISPR's novel abilities for gene manipulation, such as targeted gene disruption.

Biological intervention of CRISPR gene therapy in clinical trials.

Gene Disruption

The first clinical trial in the US using CRISPR to catalyze gene disruption for therapeutic benefit were for patients with sickle-cell anemia (SCD) and later β-thalassemia, by Vertex Pharmaceuticals and CRISPR Therapeutics. This therapy, named CTX001, increases fetal hemoglobin (HbF) levels, which can occupy one or two of four hemoglobin binding pockets on erythrocytes and thereby provides clinical benefit for major β-hemoglobin diseases such as SCD and β-thalassemia ( 103 ). The trial involved collecting autologous hematopoietic stem and progenitor cells from peripheral blood and using CRISPR/Cas9 to disrupt the intronic erythroid-specific enhancer for the BCL11A gene ( {"type":"clinical-trial","attrs":{"text":"NCT03745287","term_id":"NCT03745287"}} NCT03745287 ) as disruption of this gene increases HbF expression ( 104 – 106 ). Genetically modified hematopoietic stem cells with BCL11A disruption are delivered by IV infusion after myeloablative conditioning with busulfan to destroy unedited hematopoietic stem cells in the bone marrow. Preliminary findings from two patients receiving this treatment seem promising. One SCD patient was reported to have 46.6% HbF and 94.7% erythrocytes expressing HbF after 4 months of CTX001 transfusions and one β-thalassemia patient is expressing 10.1 g/dL HbF out of 11.9 g/dL total hemoglobin, and 99.8% erythrocytes expressing HbF after 9 months of the therapy. Results from the clinical trial that has opened for this therapy ( {"type":"clinical-trial","attrs":{"text":"NCT04208529","term_id":"NCT04208529"}} NCT04208529 ) to assess the long-term risks and benefits of CTX001 will dictate whether this approach can provide a novel therapeutic opportunity for a disease that otherwise has limited treatment options.

In vivo CRISPR Gene Therapy

While the aforementioned trials rely on ex vivo editing and subsequent therapy with modified cells, in vivo approaches have been less extensively employed. An exciting step forward with CRISPR gene therapy has been recently launched with a clinical trial using in vivo delivery of CRISPR/Cas9 for the first time in patients. While in vivo editing has been largely limited by inadequate accessibility to the target tissue, a few organs, such as the eye, are accessible. Leber congenital amaurosis (LCA) is a debilitating monogenic disease that results in childhood blindness caused by a bi-allelic loss-of-function mutation in the CEP290 gene, with no treatment options. This therapy, named EDIT-101, delivers CRISPR/Cas9 directly into the retina of LCA patients specifically with the intronic IVS26 mutation, which drives aberrant splicing resulting in a non-functional protein. The therapy uses an AAV5 vector to deliver nucleic acid instructions for Staphylococcus aureus Cas9 and two guides targeting the ends of the CEP290 locus containing the IVS26 mutation. The DSB induced by Cas9 and both guides result in either a deletion or inversion of the IVS26 intronic region, thus preventing the aberrant splicing caused by the genetic mutation and enabling subsequent translation of the functional protein ( 107 ). Potential immunotoxicity or OTEs arising from nucleic acid viral delivery will have to be closely monitored. Nonetheless, a possibly curative medicine for genetic blindness using an in vivo approach marks an important advancement for CRISPR gene therapy.

CRISPR Editing in Human Embryos and Ethical Considerations

While somatic editing for CRISPR therapy has been permitted after careful consideration, human germline editing for therapeutic intent remains highly controversial. With somatic edition, any potential risk would be contained within the individual after informed consent to partake in the therapy. Embryonic editing not only removes autonomy in the decision-making process of the later born individuals, but also allows unforeseen and permanent side effects to pass down through generations. This very power warrants proceeding with caution to prevent major setbacks as witnessed by traditional gene therapy. However, a controversial CRISPR trial in human embryos led by Jiankui He may have already breached the ethical standards set in place for such trials. This pilot study involved genetic engineering of the C-C chemokine receptor type 5 ( CCR5 ) gene in human embryos, with the intention of conferring HIV-resistance, as seen by a naturally occurring CCR5 Δ 32 mutation in a few individuals ( 108 ). However, based on the limited evidence, CRISPR/Cas9 was likely used to target this gene, but rather than replicate the naturally observed and beneficial 32-base deletion, the edits merely induced DSBs at one end of the deletion, allowing NHEJ to repair the damaged DNA while introducing random, uncharacterized mutations. Thus, it is unknown whether the resultant protein will function similarly to the naturally occurring CCR5 Δ 32 protein and confer HIV resistance. In addition, only one of the two embryos, termed with the pseudonym Nana, had successful edits in both copies of the CCR5 gene, whereas the other embryo, with pseudonym Lulu, had successful editing in only one copy. Despite these findings, both embryos were implanted back into their mother, knowing that the HIV-resistance will be questionable in Nana and non-existent in Lulu ( 109 , 110 ).

Furthermore, recent studies have shown that the mechanism for infection of some variants of the highly mutable HIV virus may heavily rely on the C-X-C chemokine receptor type 4 ( CXCR4 ) co-receptor ( 108 , 111 ). With no attempts at editing CXCR4 , this adds yet another layer of skepticism toward achieving HIV resistance by this strategy. In addition, OTEs, particularly over the lifetime of an individual, remain a major concern for applying this technology in humans. The recent advances in the editing tool to limit OTEs, such as using high fidelity Cas9 variants, has not been exploited. Furthermore, the rationale for selecting HIV prevention for the first use of CRISPR in implanted human embryos contributes to the poor risk to benefit ratio of this study, considering HIV patients can live long, healthy lives on a drug regimen. A more appropriate first attempt would have been to employ this technology for a more severe disease. For example, correction of the MYBPC3 gene is arguably a better target for embryonic gene editing, as mutations in MYBPC3 can cause hypertrophic cardiomyopathy (HCM), a heart condition responsible for most sudden cardiac deaths in people under the age of 30. Gene correction for this pathological mutation was achieved recently for the first time in the US in viable human embryos using the HDR-mediated CRISPR/Cas9 system. However, these embryos were edited for basic research purposes and not intended for implantation. In this study, sperm carrying the pathogenic MYBPC3 mutation and the CRISPR/Cas9 machinery as an RNP complex were microinjected into healthy donor oocytes arrested at MII, achieving 72.4% homozygous wildtype embryos as opposed to 47.4% in untreated embryos. The HDR-mediated gene correction was observed at considerably high frequencies with no detectable OTEs in selected blastomeres, likely owing to the direct microinjection delivery of the RNP complex in the early zygote. Interestingly, the maternal wildtype DNA was used preferentially for templated repair over the provided exogenous ssODN template ( 112 ). While evidence for gene correction was promising, NHEJ mediated DNA repair was still observed in many embryos, highlighting the need to improve HDR efficiency before clinical application can be considered. Although strategies have been developed to improve HDR, such as chemical inhibitors of NHEJ ( 77 – 79 ), such techniques may have varying outcomes in embryonic cells and side effects that may arise from treatment needs to be investigated. Germline gene editing will remain to be ethically unfavorable at its current state and its discussions may not be considered until sufficient long-term studies of the ongoing somatic CRISPR therapy clinical trials are evaluated.

Potential for CRISPR Therapeutics During COVID-19 Pandemic

The rapidly advancing CRISPR technology may provide aid during our rapidly evolving times. The recent outbreak of a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic ( 113 ). These pressing times call for an urgent response to develop quick and efficient testing tools and treatment options for coronavirus disease 2019 (COVID-19) patients. Currently available methods for testing are relatively time consuming with suboptimal accuracy and sensitivity ( 114 ). The two predominant testing methods are molecular testing or serological testing. The US Centers for Disease Control and Prevention (CDC) has developed a real-time RT-PCR assay for molecular testing for the presence of viral RNA to detect COVID-19 ( 115 ). However, this assay has a roughly ~30% false negative rate ( 116 , 117 ) with the turnaround time of several hours to >24 h. Serological testing methods are much more rapid but lack the ability to detect acute respiratory infection since antibodies used to detect infection can take several days or weeks to develop.

Recently, a CRISPR Cas12-based assay named SARS-CoV-2 DETECTR has been developed for detection of COVID-19 with a short turnaround time of about 40 min and a 95% reported accuracy. The assay involves RNA extraction followed by reverse transcription and simultaneous isothermal amplification using the RT-LAMP method. Cas12 and a guide RNA against regions of the N (nucleoprotein) gene and E (envelope) gene of SARS-CoV-2 are then targeted, which can be visualized by cleavage of a fluorescent reporter molecule. The assay also includes a laminar flow strip for a visual readout, where a single band close to where the sample was applied indicates a negative test and 2 higher bands or a single higher band would indicate cleavage of the fluorescent probe and hence positive for SARS-CoV-2 ( 118 ).

In addition to CRISPR's diagnostic utility, CRISPR may provide therapeutic options for COVID-19 patients. The recently discovered Cas13 is an RNA-guided RNA-targeting endonuclease may serve as a potential therapeutic tool against COVID-19. PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells) has been developed, which utilizes the Ruminococcus flavefaciens derived VI-D CRISPR-Cas13d variant, selected for its small size facilitating easier packaging in viral vehicles, high specificity, and strong catalytic activity in human cells. This technique was developed to simultaneously target multiple regions for RNA degradation, opening the door for a much-needed pan-coronavirus targeting strategy, given the evidence suggesting relatively high mutation and recombination rates of SARS-CoV-2 ( 119 ). With these advances, the CRISPR/Cas machinery may again be implemented to serve its original purpose as a virus-battling system to provide aid during this pandemic.

The birth of gene therapy as a therapeutic avenue began with the repurposing of viruses for transgene delivery to patients with genetic diseases. Gene therapy enjoyed an initial phase of excitement, until the recognition of immediate and delayed adverse effects resulted in death and caused a major setback. More recently, the discovery and development of CRISPR/Cas9 has re-opened a door for gene therapy and changed the way scientists can approach a genetic aberration—by fixing a non-functional gene rather than replacing it entirely, or by disrupting an aberrant pathogenic gene. CRISPR/Cas9 provides extensive opportunities for programmable gene editing and can become a powerful asset for modern medicine. However, lessons learned from traditional gene therapy should prompt greater caution in moving forward with CRISPR systems to avoid adverse events and setbacks to the development of what may be a unique clinically beneficial technology. A failure to take these lessons into account may provoke further backlash against CRISPR/Cas9 development and slow down progression toward attaining potentially curative gene editing technologies.

Although CRISPR editing in humans remains a highly debated and controversial topic, a few Regulatory Affairs Certification (RAC)-reviewed and FDA-approved CRISPR gene therapy trials have opened after thorough consideration of the risk to benefit ratios. These first few approved trials, currently in Phase I/II, are only for patients with severe diseases, such as cancers or debilitating monogenic diseases. The outcomes of these trials will dictate how rapidly we consider using this system to treat less severe diseases, as the risks of the technology are better understood. A concern remains whether normalizing CRISPR/Cas9 editing for less debilitating diseases may act as a gateway for human genome editing for non-medical purposes, such as altering genes in embryos to create offspring with certain aesthetic traits. This fear of unnatural selection for unethical reasons has likely become more tangible in the public's view with the strong media attention of the edited “CRISPR babies.” The lasting effects of that trial and outcomes of the approved clinical trials will greatly influence CRISPR's future in gene therapy and begin to answer the key questions we must consider as we further explore this technology. These key questions include how to avoid the mistakes of the past, who should decide CRISPR's therapeutic future, and how the ethical boundaries of its applications should best be drawn.

Author Contributions

FU researched and drafted the article. TS and CR supervised the content. All authors wrote, reviewed, and edited the manuscript before submission.

Conflict of Interest

CR has consulted regarding oncology drug development with AbbVie, Amgen, Ascentage, Astra Zeneca, Celgene, Daiichi Sankyo, Genentech/Roche, Ipsen, Loxo, and Pharmar, and is on the scientific advisory boards of Harpoon Therapeutics and Bridge Medicines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors would like to thank Ms. Emily Costa, Dr. Alvaro Quintanal Villalonga, and Dr. Rebecca Caesar for their excellent assistance with editing the review.

Funding. This work was supported by grants from the US National Institutes of Health, including U24CA213274 and R01CA197936 (CR); Parker Institute of Cancer Immunotherapy grant (TS).

Gene therapy: principles, challenges and use in clinical practice

• review article
• Open access
• Published: 07 May 2024

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• Cihan Ay MD   ORCID: orcid.org/0000-0003-2607-9717 1 &
• Andreas Reinisch MD PhD 2  

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Introduction

Gene therapy is an emerging topic in medicine. The first products have already been licensed in the European Union for the treatment of immune deficiency, spinal muscular atrophy, hemophilia, retinal dystrophy, a rare neurotransmitter disorder and some hematological cancers, while many more are being assessed in preclinical and clinical trials.

The purpose of this review is to provide an overview of the core principles of gene therapy along with information on challenges and risks. Benefits, adverse effects and potential risks are illustrated based on the examples of hemophilia and spinal muscular atrophy.

At present, in-vitro and in-vivo gene addition or gene augmentation is the most commonly established type of gene therapy. More recently, more sophisticated and precise approaches such as in situ gene editing have moved into focus. However, all types of gene therapy require long-term observation of treated patients to ensure safety, efficacy, predictability and durability. Important safety concerns include immune reactions to the vector, the foreign DNA or the new protein resulting from gene therapy, and a remaining low cancer risk based on insertional mutagenesis. Ethical and regulatory issues need to be addressed, and new reimbursement models are called for to ease the financial burden that this new treatment poses for the health care system.

Gene therapy holds great promise for considerable improvement or even cure of genetic diseases with serious clinical consequences. However, a number of questions and issues need to be clarified to ensure broad accessibility of safe and efficacious products.

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Already in 1972, Friedmann and Roblin hypothesized that genetic modification might be the way to cure hereditary diseases [ 1 ]. Following many years of scientific groundwork and technical advancements, the first clinical gene therapy studies started in the early 1990s [ 2 ]. Over the years, several major setbacks, including the tragic death of a patient treated in a gene therapy trial in 1999 and several cases of unintended insertional mutagenesis and development of acute leukemia, slowed the development [ 3 , 4 , 5 , 6 , 7 ]. The 18-year-old patient who died in 1999 had partial ornithine transcarbamoylase (OTC) deficiency, a genetically determined metabolic disorder of the urea cycle, and received an infusion of corrective OTC gene encased in a recombinant adenoviral vector [ 8 ]. A severe immune reaction evoked by the adenoviral vector led to his death four days after the administration. This case highlighted the potential of the vector itself to pose a risk, adequate training of the health care staff and the implementation of basic operating procedures, among others.

The lessons learned from these events enabled continuous improvements, and the unprecedented results that are achievable with gene therapy led to the development of a myriad of new products currently tested in clinical trials for a broad range of indications [ 9 ]. Today, gene therapies have emerged as promising treatment options and are rapidly entering the treatment landscape of various inherited and acquired diseases including immune disorders, neurodegenerative diseases, hemophilia, ocular diseases, hemoglobinopathies, or cancer. Some gene therapies have already been approved for clinical use, and many more are being developed at increasing speed.

Although continuous collection of additional long-term safety data will be necessary in the future, the growing importance of gene therapy is beyond doubt and calls for appropriate knowledge among health care professionals. There is currently a high unmet need for education, as revealed by a survey conducted among hospital physicians in Austria [ 10 ]. To address this knowledge gap, this review summarizes core principles, benefits, potential risks and challenges of gene therapy, with a particular focus on hemophilia and spinal muscular atrophy, and discusses future perspectives.

Basic principles of gene therapy

Methods and techniques of gene therapy: gene addition/augmentation vs. gene suppression.

Gene therapy is the transfer of genetic material to a patient to treat or potentially even cure a disease. There are various approaches of gene therapy. Most currently used gene therapy products attempt to replace the function of a defective gene with that of a healthy gene. The genetic material (aka transgene) should ideally be delivered to the physiologically relevant target tissue where it is expressed at a physiologically meaningful level and in a stable manner. Interference of the gene or its protein products with the integrity of the target cells must be avoided [ 11 ].

This addition or substitution of genes with loss-of-function defects (Fig.  1 a) is called gene addition/augmentation [ 2 ]. In case of gene augmentation, the newly transferred functional copy of a gene is present in the cell nucleus together with the defective gene.

Types of gene therapy: gene augmentation, gene suppression, and genome editing (according to Anguela XM and High KA, and Porteus MH) [ 2 , 12 ]. Foot note: NHEJ  non-homologous end joining; repair of double-strand breaks via direct ligation of the break ends without a homologous template

A different approach is required when the disease is caused by gain-of-function defects. Suppression of gain of function can be achieved by the import of inhibitory sequences (i.e., microRNAs, short hairpin RNAs) into target cells (Fig.  1 b).

Genome editing

More recently, with the discovery of novel tools that can precisely target and manipulate DNA, in situ repair of genetic defects has become possible. This approach is called genome editing and allows for the correction of genetic defects with single base-pair precision (Fig.  1 c; [ 2 , 12 ]). Genome editing mostly relies on the site-specific introduction of a double-strand break (DSB) into DNA. The nuclease-induced site-specific DSB in the genome stimulates active endogenous repair mechanisms. The two best understood repair mechanisms are non-homologous end-joining (NHEJ) and homology-directed repair (HDR). In NHEJ, the DSB is fixed simply by joining the two ends of the broken DNA. This mechanism is relatively error-prone and frequently leads to insertions and deletions (indels) that can result in the functional loss of a gene. In stark contrast, HDR—the naturally occurring recombination mechanisms observed in mammalian cells—involves the use of the sister chromatide as a template for a “copy and paste” process known as homologous recombination. However, for genome editing, a cell can be tricked, and a DNA donor repair template can be introduced into the cell that will be used for homologous recombination instead of the sister chromatide. Importantly, this DNA repair template can be engineered to correct a mutation or even integrate additional genetic material.

CRISPR-Cas9 is currently the platform that is used most frequently for the introduction of DNA DSBs. However, other nucleases such as zinc-finger nucleases, transcription activator-like effector nucleases and homing endonucleases-meganucleases offer comparable precision.

Genome editing can be performed in cells both outside and inside of the body ( ex vivo and in vivo , see below) [ 12 ]. Clinical trials are currently assessing the utility of genome editing in the correction of monogenic diseases and cell-based regenerative medicine; also, enhancement of chimeric antigen receptor (CAR) T cell therapy is investigated. In principle, genome editing has the potential to halt the progression of most monogenic diseases, provided that patients are treated before irreversible damage has occurred. The first CRISPR-based gene therapy, which has been approved only recently in the European Union is exagamglogene autotemcel for the treatment of transfusion-dependent β‑thalassemia and severe sickle cell disease [ 13 ].

Methods of gene transfer: ex vivo vs. in vivo gene therapy

Ex vivo gene therapy requires the extraction of cells from the patient [ 14 ]. After the successful introduction of a transgene or successful gene editing, the cells are re-administered to the patient. Ex vivo gene therapies commonly use cells of hematopoietic origin such as hematopoietic stem cells or T cells, and are being developed for the treatment of inherited immune disorders, neurodegenerative diseases (e.g., X‑linked adrenoleukodystrophy, metachromatic leukodystrophy), β‑hemoglobinopathies (e.g., β‑thalassemia, sickle cell disease), and cancer.

Today, gene therapy for cancer has mainly been established in the form of CAR T cell therapy. CARs endow T cells with the ability to target antigens expressed on the surface of tumor cells. CD19, an antigen present in most B cell malignancies, constitutes the classical target, with several products having been approved to date. However, CAR T cell applications are extending to other hematologic malignancies as well as solid tumors.

In contrast to ex vivo gene therapy, in vivo gene therapy relies on the administration of a vector that carries and delivers the transgene to a target tissue (e.g., liver, neurons, muscle). Most current in vivo gene therapies target the patient’s liver and can be applied intravenously [ 15 ]. In vivo gene therapy usually does not require conditioning prior to administration; thus, prolonged hospital stays are avoided, and the treatment can frequently even be conducted in the outpatient setting. Moreover, this approach is attractive as it dispenses with the need for elaborate steps involved in ex vivo treatment including cell collection from the patient and manipulation in a specialized facility before re-administration [ 16 ]. However, the feasibility of in vivo administration depends on tissue-specific targeting or local delivery and/or target-cell-specific gene expression.

In vivo gene therapy primarily focuses on rare monogenic disorders caused by loss-of-function or toxic gain-of-function mutations. Among others, in vivo gene therapy is currently being evaluated or has already been approved in the treatment of hemophilia A and B, neuromuscular disorders (e.g., spinal muscular atrophy, Duchenne muscular dystrophy) and various types of inherited blindness (e.g., RPE65 mutation-associated retinal dystrophy, achromatopsia, choroideremia, Leber’s hereditary optic neuropathy, X‑linked retinoschisis, and X-linked retinitis pigmentosa). In the setting of central nervous system diseases, targeting a sufficient number of cells to achieve an adequate level of gene modification is challenging [ 2 ].

Vectors are vehicles that can carry genetic material and introduce it into target cells. For gene transfer, mainly naturally occurring viruses are genetically modified for the purpose of transferring and expressing a transgene. In viral vectors, the viral genome is replaced by the gene therapy transgene. One fundamental distinction between the viruses used for gene therapy is their inherent capacity to integrate into the host DNA. Therefore, viral vectors can broadly be classified as either integrating or non-integrating [ 17 , 18 ].

Integrating viral vectors are introduced into cells with the aim of stably incorporating therapeutic genes into the genome, thus allowing the cells to pass the transgene onto every daughter cell. These vectors, which are typically derived from retro- and lentiviruses, are frequently employed for ex vivo gene therapy.

With non-integrating viral vectors, on the other hand, the transferred DNA is stabilized extrachromosomally as an episome. Since the transgene is usually not integrated into the genome, it needs to be delivered to long-lived, non-dividing, post-mitotic cells where it will be expressed for the life of the target cell only. Episomes are stable in non-dividing cells for long periods and provide sustained transgene expression [ 19 , 20 ]. On the downside, transgene expression may be lost over time upon cell proliferation due to the lack of vector genome replication with cell division [ 21 ]. Non-integrating vectors are typically used for in vivo gene therapy.

Recombinant adeno-associated viral (rAAV) vectors have emerged as the platform of choice for in vivo gene therapies due to their advantages of relatively low immunogenicity, targeted gene delivery into a range of tissues, and long-term expression of the transgene [ 22 , 23 , 24 ]. AAV is a very small ( parvovirus) single-stranded DNA virus that is non-pathogenic and naturally replication-defective. Wild-type AAV requires the presence of another virus, such as an adenovirus, to replicate [ 25 ]. In the process of engineering, all viral coding sequences including the rep and cap genes that are responsible for replication and the structure of the viral capsid are replaced with a gene expression cassette of interest. This includes not only the therapeutic gene but also other transcriptional regulatory elements such as a promoter sequence that facilitates transgene expression within specific cell types [ 26 ]. rAAV vectors have tropism for specific tissues depending on their serotype, with serotypes ranging from AAV1 to AAV13 [ 27 ]. Features of different vectors are discussed in Table  1 [ 2 , 28 ].

For the large-scale production of rAAV vectors, platforms based on human embryonic kidney cells (HEK) or the insect cell line Spodoptera frugiperda (SF9) with recombinant baculoviruses have been widely employed [ 22 ]. rAAVs are administered via a single infusion either intravenously or locally. If given intravenously, the vector will transduce the target cell depending on its tissue tropism. Once bound to the cell via receptors, the virus gets endocytosed. After escaping from the endosome, rAAV particles enter the cell nucleus, the viral capsid gets uncoated, and after a second strand synthesis of the transgene, the host cell’s endogenous transcription and translation machinery is used for the production of a functional protein. Based on the fact that rAAVs integrate into the host genome at very low frequencies, rAAV is considered to bear only low risk of genotoxicity [ 29 , 30 , 31 , 32 , 33 ]. In clinical studies investigating valoctocogene roxaparvovec and etranacogene dezaparvovec that have been licensed for the treatment of hemophilia A and B, respectively, transgene DNA was temporarily detected in semen; therefore, barrier contraception is recommended for 6 and 12 months after the administration of valoctocogene roxaparvovec and etranacogene dezaparvovec, respectively, in patients with reproductive potential [ 34 , 35 ]. Moreover, treated patients should not donate semen, blood, organs, tissues or cells for transplantation.

Risks of gene therapy

Depending on the type of gene therapy ( ex vivo vs. in vivo , integrating vs. non-integrating vectors), several safety-related issues need to be taken into consideration and should be discussed with the potential patient.

Integrating vectors such as retro- and lentiviruses that are primarily used for ex vivo gene therapy bear the risk of insertional mutagenesis due to their semi-random integration into the DNA. This can potentially induce the activation of an oncogene or the disruption of a tumor suppressor gene, thereby leading to the formation of cancer [ 6 , 36 , 37 , 38 ]. Unfortunately, T cell leukemia developed in some of the early trials using γ‑retroviral vectors for severe combined immunodeficiencies (SCID) [ 39 ]. Over time, the risk of insertional mutagenesis has been reduced by the development of safer vectors [ 18 ]. Compared to γ‑retroviral vectors, lentiviral vectors have a safer integration pattern and higher transduction efficiencies. However, clinical-scale production of lentiviral vectors is challenging. Nevertheless, specific surveillance and long-term follow-up is necessary. In the future, such unintentional detrimental integration events might be avoided by using the very precise genome editing technology [ 18 ].

In contrast to integrating viral vectors that are primarily used for ex vivo gene therapy applications, non-integrating viral vectors are mainly used for in vivo gene therapy. These have only minimal rates of integration into the donor DNA and consequently confer a very low probability of causing insertional mutagenesis and cancer. Hemophilic dogs treated with AAV gene therapy had low but detectable levels of AAV integration into the genomic DNA and did not show any evidence of tumor formation after 10 years of follow-up [ 40 ]. Studies in neonatal mice implicated that pathogenic AAV integration events might actively contribute to hepatocellular cancer development, although potential genotoxic events are highly dependent on factors including AAV integration preferences, vector design, vector dose and, in particular, recipient age at AAV injection [ 41 , 42 , 43 ].

Since non-integrating vectors are applied in vivo , they carry the risk of evoking immune responses that are potentially life-threatening or might impair the long-term efficacy of treatment. Immune responses and related adverse events seem to be directly associated with the vector doses applied [ 44 , 45 ]. Uncontrolled immune responses are the main culprit with regard to most severe adverse events linked to AAV gene transfer, including fatal hepatotoxicity, dorsal root ganglia toxicity, and myocarditis. Notably, the human body contains immune-privileged sites (e.g., the central nervous system) and immunosuppressive microenvironments (e.g., the liver) where AAV vectors are less likely to trigger strong responses than at other sites such as the circulation or the muscle [ 46 ].

Uncontrolled innate immunes responses such as overactivation of the complement pathway with subsequent induction of thrombotic microangiopathy have been described following AAV gene therapy. Thrombotic microangiopathy is a hematologic emergency situation caused by microscopic blood clots in the capillaries and small blood vessels, leading to organ damage, anemia and low platelet counts [ 47 ]. Also, the adaptive immune system can cause dangerous side effects via CD8+ cytotoxic T‑cell responses, such as T‑cell mediated hepatotoxicities associated with inflammatory reactions that have been observed in AAV9 vector therapy for spinal muscular atrophy.

Immune responses to vectors can be mitigated by the administration of immunomodulatory drugs such as corticosteroids [ 18 ]. However, immune system-mediated toxicity is still a challenge for successful gene transfer using AAV vectors, particularly in settings in which the treatment of the targeted genetic disease requires high doses [ 48 ].

Approved therapies and fields of investigation

A number of gene therapy products have been licensed over the last seven years in Europe, the United States and other countries. Currently, a total of six CAR T cell products have received approval in Europe. Tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel and lisocabtagene maraleucel are used for the treatment of patients with B‑cell malignancies (e.g., lymphoma); all of these target the CD19 antigen [ 49 , 50 , 51 , 52 ]. Tisagenlecleucel is also indicated for acute lymphoblastic leukemia [ 49 ]. The BCMA-directed therapies idecabtagene vicleucel and ciltacabtagene autoleucel have been licensed for the treatment of multiple myeloma [ 53 , 54 ]. Talimogene laherparepvec is a modified oncolytic herpes virus that is used as an intralesional cancer immunotherapy for advanced melanoma [ 55 ].

In addition, at the time of the publication of this review, gene therapies are available in Europe for serious monogenic disorders including severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID; autologous CD34+ cells transduced with a retroviral vector that encodes for the human ADA complementary DNA sequence), biallelic RPE65 mutation-associated retinal dystrophy (voretigene neparvovec), aromatic L‑amino acid decarboxylase (AADC) deficiency (eladocagene exuparvovec), metachromatic leukodystrophy (atidarsagene autotemcel), spinal muscular atrophy (onasemnogene abeparvovec), hemophilia A (valoctocogene roxaparvovec) and B (etranacogene dezaparvovec) and β‑thalassemia as well as sickle cell disease [ 13 , 34 , 35 , 56 , 57 , 58 , 59 , 60 ].

A multitude of trial programs is currently evaluating gene therapies in a broad range of diseases. Approximately 1500 products are being tested in the pre-clinical setting and in more than 500 clinical studies. In addition to the mentioned indications, gene therapy is being assessed in inherited metabolic diseases such as ornithine transcarbamylase deficiency (NCT02991144), homozygous familial hypercholesterolemia (NCT02651675) and mucopolysaccharidosis type VI (NCT03173521), in age-related macular degeneration (NCT01024998, NCT01301443, NCT01494805, NCT03066258) and previously untreatable disorders like Huntington’s disease (NCT03761849), among many others. As an example, achievements and limitations of established gene therapies are delineated below for hemophilia and spinal muscular atrophy.

Hemophilia a and b

Hemophilia, an X‑linked recessive bleeding disorder, is caused by a deficiency of coagulation factor VIII (hemophilia A) or IX (hemophilia B) due to mutations in the genes encoding for these factors. Several characteristics make hemophilia A and B an ideal target for gene therapy: this is a monogenic, recessive disease which results in a large range of affected protein levels [ 61 , 62 , 63 ]. Moreover, the bleeding phenotype is responsive to increases of factor levels, and their measurement provides monitoring of the treatment efficacy. While FVIII is synthesized in the sinusoidal endothelial cells of the liver, FIX synthesis takes place in the hepatocytes [ 63 , 64 ]. The majority of defects of the F8 gene are caused by intron 22 inversions; in the F9 gene, missense mutations are mainly responsible for the absence or dysfunction of the clotting factor [ 65 , 66 ].

In patients with hemophilia, FVIII or FIX deficiency leads to bleeding into joints, muscles and soft tissues, eventually giving rise to joint damage, disability and chronic pain as the most common consequences [ 61 ]. The traditional treatment consists of intravenous replacement of coagulation factor concentrates at regular intervals, given the relatively short half-life of these factors. This puts a considerable burden on patients and care givers. Furthermore, persons with hemophilia may develop inhibitory antibodies that diminish the efficacy of factor replacement. Despite regular prophylaxis, the risk of arthropathy is not completely reduced with the current treatment options. Moreover, the treatment confers a significant cost burden, and access to factor products is limited in many countries.

Valoctocogene roxaparvovec was the first gene therapy to be licensed for the treatment of hemophilia A and became available in August 2022 in the European Union [ 34 ]. Similarly, etranacogene dezaparvovec was approved as the first gene therapy for hemophilia B in February 2023 [ 35 ].

Gene therapy for hemophilia is liver-directed as the vectors target hepatocytes, which act as protein factories to release the transgene product into the circulation. AAV vectors with the serotype 5 are used for both currently approved liver-directed therapies. This treatment is expected to transform severe disease phenotypes into mild or normal phenotypes based on sustained elevation of clotting factor levels [ 2 , 63 , 67 , 68 ]. The continuous expression of coagulation factors provides protection from bleeding, renders prophylaxis at regular intervals unnecessary and contributes to increased quality of life.

While hemophilia A and B show similar clinical symptoms, their molecular bases differ. As FVIII complementary DNA is larger than FIX complementary DNA (approximately 9 kb vs. 1.5 kb), modification is required to enable packaging of the F8 transgene into the recombinant AAV5 (rAAV5) vector [ 65 , 66 , 69 ] that has limited packaging capacity of approximately 4.7 kb (Fig.  2 ). To fit the F8 transgene into AAV, the large B‑domain of F8 is deleted, resulting in a length of approximately 5 kb. For the F9 transgene, a naturally occurring but more active variant of FIX that was initially described in a family in Padua (i.e., the Padua variant) is often used [ 70 ]. The therapy is administered as a single intravenous infusion, with dosing based on body weight. Following the administration, patients may develop a mild viral syndrome consisting of transient fever, myalgia, and malaise [ 71 , 72 , 73 ].

Structure of adeno-associated viral vectors for the treatment of hemophilia ( a ) and ( b ). Foot note: ITR  inverted terminal repeat; pA  polyadenylation signal

AAVs naturally infect humans, and upon infection the human immune system develops neutralizing antibodies that are a particular challenge for AAV-based gene therapy approaches. These pre-existing neutralizing anti-AAV antibodies impede gene transfer by inhibiting the transduction of target cells by the AAV-based vector [ 74 ]. Measurable antibodies to different AAV serotypes have been found in approximately 30–60% of the population [ 75 ]. Prior to treatment with the gene therapy product approved for hemophilia, the levels of neutralizing antibodies need to be assessed. Only patients without antibodies according to a validated assay are eligible for the administration of valoctocogene roxaparvovec [ 34 ]. With respect to etranacogene dezaparvovec, patients with pre-existing anti-AAV5 antibodies were not excluded from the phase III trial. Trials results showed that gene therapy can be successful even in the presence of low titers of pre-existing neutralizing antibodies; however, the titer should not exceed 1:678 according to the specific assay employed for etranacogene dezaparvovec [ 35 ].

Accurate and robust detection of neutralizing anti-AAV antibodies is important but not easy to achieve as the required assays have not been established in clinical routine yet. Furthermore, no universal method has been implemented to reliably measure the amount of clinically relevant antibody levels [ 76 ]. Transduction inhibition assays and total antibody assays are used, although meaningful comparisons across assays are nearly impossible due to the lack of standardization. The limited availability of head-to-head studies that align assay results with clinical outcomes renders the interpretation and implementation of screening titer cutoffs difficult [ 77 ].

Another issue that requires attention is the emergence of potential immune responses against capsid proteins or even the transgene and its products that can lead to rejection of the transduced cells [ 78 , 79 , 80 ]. In a high number of patients, liver-directed gene therapy for hemophilia led to modest increases in the liver transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST) [ 78 ]. Although all hemophilia gene therapy clinical trials have shown transaminitis, this was more frequently seen in patients receiving hemophilia A gene therapy than in those undergoing hemophilia B gene therapy [ 81 , 82 ]. In the majority of cases, the reported elevations in ALT levels showed a 1.5- to 2‑fold peak above the upper limit of normal. Unfortunately, the mechanisms responsible for ALT elevation potentially reflecting liver damage have not been fully unraveled to date, but cytotoxic T‑cell attacks against transduced cells and/or cellular stress induced by the accumulation of misfolded protein in the endoplasmic reticulum are suspected [ 20 , 23 , 83 ]. Transaminitis mainly occurred within the first 12 weeks after vector infusion and either preceded a loss of transgene expression or coincided with it [ 78 , 84 ]. In clinical studies, immunosuppression with corticosteroids was initiated with the aim of dampening the immune system and thereby preserving the expression of the gene therapy product [ 80 ].

During the first weeks and months following administration of gene therapy, close clinical and laboratory monitoring is mandatory [ 66 ]. If the gene transfer is successful, the need for exogenous administration of coagulation factor products generally declines considerably until the endogenous factor production has sufficiently increased to render factor replacement therapy unnecessary [ 85 , 86 ]. However, patients need to be aware of considerable inter-patient outcome variability that has been observed in clinical trials. Additionally, each gene therapy product has its unique features, including vector design and vector dose, AAV serotype and the production platform used for manufacturing. Patient variables include previous AAV exposure, patient-specific antigen processing, and hepatic health prior to gene therapy [ 44 , 71 , 85 , 87 , 88 , 89 ]. Further research and long-term observation is needed to gain additional insights, especially with respect to safety as well as predictability and durability of factor expression.

• Spinal muscular atrophy

Loss-of-function mutations in the survival motor neuron 1 ( SMN1 ) gene give rise to spinal muscular atrophy (SMA) [ 90 ]. This autosomal recessive disease is characterized by the degeneration of alpha motor neurons located in the spinal cord. Progressive muscle weakness, paralysis, loss of bulbar function and death from respiratory complications occur at around 2 years of age in most patients [ 91 , 92 ]. Infantile-onset (type 1) SMA is the most severe and most common subtype of SMA [ 93 ]. It usually manifests before the age of 6 months and is the most common genetic cause of death in infants; however, symptoms may already be present at birth.

The antisense oligonucleotide drug nusinersen has revolutionized the treatment of patients with SMA. It targets the SMN2 gene, which is a nearly identical copy of the SMN1 gene, and produces functional SMN protein, although only a fraction of the amount obtained from the intact SMN1 gene, and thus cannot compensate for the loss of SMN1 [ 94 , 95 ]. By modulating alternative mRNA splicing of the SMN2 gene in spinal motor neurons, nusinersen induces higher expression of SMN2 , thereby better compensating for the SMN1 loss. However, this treatment involves repeated intrathecal administration (i.e., direct injection into cerebrospinal fluid) with up to seven injections during the first year followed by maintenance doses every 4 months. The treatment costs are substantial, and patients with advanced disease still rely on assisted respiration using non-invasive ventilation [ 96 ]. In addition, the oral SMN2 pre-mRNA splicing modifier risdiplam has been approved for the treatment of patients with 5q-autosomal recessive SMA with a clinical diagnosis of SMA types 1, 2, or 3, or with one to four copies of the SMN2 gene [ 97 ].

The first gene therapy for patients with SMA is onasemnogene abeparvovec, which was approved in the European Union in 2020. It is indicated in patients with SMA linked to chromosome 5q, a biallelic mutation in the SMN1 gene and clinically apparent SMA type 1, or 5q-associated SMA with a biallelic mutation in the SMN1 gene and up to three copies of the SMN2 gene [ 60 ]. Onasemnogene abeparvovec is an AAV-based gene therapy that is administered as a one-time intravenous infusion. The AAV vector serotype 9 (AAV9) delivers a fully functional copy of the SMN gene into the target motor neuron cells, leading to expression of the SMN protein.

SMA patients treated with onasemnogene abeparvovec show improvements in muscle movement and function, significant improvement in their ability to reach developmental motor milestones, and survival prolongation. Long-term study results suggest evidence of sustained clinical efficacy. The phase I START study included symptomatic infants with SMA type 1 and two copies of the SMN2 gene [ 98 ]. After a median of 5.2 years post gene therapy, all 10 patients in the therapeutic-dose cohort remained alive and without the need for permanent ventilation. All of them had maintained previously acquired motor milestones, and two had achieved the milestone of standing without assistance. The phase III SPR1NT trial evaluated onasemnogene abeparvovec in pre-symptomatic children with biallelic SMN1 mutations treated within 6 weeks after birth [ 99 ]. Among 15 children with three SMN2 copies, all were able to stand independently before 24 months, and 14 walked independently. All of them survived without permanent ventilation at 14 months.

Mandatory assessments prior to the treatment include measurement of pre-existing AAV9 antibodies using a validated assay and liver function tests. The most common side effects of onasemnogene abeparvovec comprise elevation of liver enzymes and vomiting. As acute hepatic failure with a fatal outcome has been reported, it is recommended to monitor the liver function regularly for at least 3 months after treatment [ 100 ]. Immune responses to the vector are assumed to be the cause of hepatotoxicity; therefore, a prophylactic corticosteroid regimen needs to be administered. Moreover, available data suggest that overexpression of the SMN protein, especially in the sensorimotor circuit, might lead to gain of toxic function [ 101 ]. A long-term follow-up study (NCT03421977) of the completed phase 1 study (NCT02122952) is assessing safety for up to 15 years, with final results expected for December 2033.

Challenges & perspectives

Gene therapy has opened new doors in the treatment of a range of serious and debilitating diseases. However, many remaining challenges need to be fully addressed before gene therapy can become a routine treatment for monogenic diseases [ 18 , 102 ]. These include mainly aspects related to safety, predictability, and the durability of the gene therapy outcomes. For in vivo gene therapy, better understanding of immune responses is needed, and systematic long-term efficacy and safety assessment of every treated patient will be essential. Moreover, manufacturing and regulatory challenges need to be solved to make gene therapies broadly accessible. Since gene therapy requires well-trained personnel working at specialized facilities, the number of centers providing these therapies will be limited [ 2 ].

Finally, a societal consensus needs to be reached regarding disputed issues such as the very high financial burden [ 18 ]. One-time gene therapies tend to be extremely expensive up-front, although cost-benefit analyses that take patient quality of life and lifelong medical costs of currently available treatments into account may provide justification for the use of gene therapy products [ 103 ]. In addition, treatment options have been completely absent for a range of serious diseases to date. Nevertheless, keeping the expenses at a reasonable level will be important to improve equality of access. Dedicated funding programs can help to lower the financial burden. Negotiations with health insurances and government agencies might result in the development of new models for reimbursement.

Finally, to implement gene therapy in clinical practice special logistics and a multidisciplinary approach will be required. Various organization in the field of hemophilia propose new delivery models, such as the hub and spoke model to gain access to gene therapy for patients (summarized in [ 104 ]). In such a care delivery model a close collaboration and communication between the hub center, which is responsible for administration of gene therapy and the spoke center (i.e. the referral center) is needed to cover the management of the complex patient journey form initial discussion to long-term follow up.

Gene therapies are promising and offer enormous potential with respect to finally achieving cure of many serious hereditary and non-hereditary diseases. In 2024, the history of their development already spans decades, although in clinical terms, it appears that the journey has barely begun. Study results obtained with approved gene therapies have proven the principle of gene therapy for clinical use. Nevertheless, to make this new treatment approach broadly available, very demanding challenges regarding both medical and regulatory/financial issues need to be addressed. Long-term safety, clinical efficacy and advantages over standard treatment options must be clearly demonstrated to justify the high-cost burden. Also, ethical discussions are needed to determine an acceptable framework for these new procedures. With numerous trials investigating gene therapies in various indications ongoing, patients with devastating diseases can now hope for new and unprecedented treatment options.

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Gene Therapy: Risks and Benefits Research Paper

Introduction, summary of the mechanisms of gene therapy, discussion of paper and critique of the methods used, argument and future experiments proposed, works cited.

Gene therapy is the “insertion or removal of genes which can also be alternated within the cell or tissues of an organism for purposes of treating diseases” (Cross & Burmester). All over the world, “the technique is best known for the correction of defective genes so as to treat diseases; the most common procedural form of gene therapy involves the insertion of the functional gene in order to replace the mutated gene within an organism” (Cross & Burmester).

Over the recent past use of gene therapy has revolutionized the treatment approaches, and research on this subject is justified because of the great potential that this technique offers. Similarly, further research will also shed light on possible risk factors that are associated with this method of treatment. The purpose of this paper is to undertake a cost-benefit analysis of gene therapy as a treatment option.

Germ-line gene therapy

Under normal circumstances, germ-line gene therapy procedures involve alternating and replacing all defective genes within the body of an organism (Walesgenepark.co.uk). In this case, functional genes are isolated and inserted into the interior lobes of the reproductive tissues or cells of the relevant organism. Considerably, this therapy operates on the basis that fresh and healthy genes get inoculated into the gametes of an organism in order to reduce the risks of later transfer of the defective genes to the next generation of the organism (Walesgenepark.co.uk).

Moreover, there is the involvement of complete alteration of the genetic makeup of early-stage blastomere so as to enhance changes of the genetic codes which can be passed on from generation to generation. Secondly, germ-line therapy also involves alteration of the gametes before fertilization since, after fertilization, the characteristics are passed on to the offspring’s (Walesgenepark.co.uk).

Somatic gene therapy

Generally, “somatic gene therapy involves the process of alteration of the genetic makeup of somatic cells (i.e., all body cells except sex cells) of an individual” (Rothchild, Laura & Lauren). Contrary to germ-line therapy, there is no transfer of these cellular changes to the next generation of the organism, simply because they are neither sex cells nor gametes which fuse during fertilization (Rothchild, Laura & Lauren). The main focus on the process involves changing the arrangements of the genetic codes of the specific cells using either an in-vitro or ex-vivo DNA delivery system. This technique in today’s life can be employed medically in treatments of a variety of diseases, including; hemophilia, muscular dystrophy, among many others; currently, it is used for cancer treatments (Rothchild et al.).

Telomerase inhibition strategies

In humans, it’s evident that the telomerase RNA abbreviated (hTR) plays an important role in anticancer therapy. This hTR can be used as an anticancer either individually or in combination with another human telomerase reverse transcriptase (hTERT) (Li, Li, Yao, Geng, Xie, Feng, Zhang, Kong, Xue, Cheng, Zhou & Xiao 4). Current findings indicate that when these two agents combine with the recombinant adenovirus, another nucleotide called small-interfering RNA (siRNA) is formed (Li et al. 4).

Furthermore, it has been established that the levels of telomerase activities together with mRNA, hTR are greatly reduced by the activities of the recombinant adenovirus resulting in inhibition of Xenograft tumor growth (Li et al. 4). This implies that siRNA, which is specifically expressed in recombinant adenoviruses, is the best tool to be used as an anticancer and also in the treatment of oral squamous cell carcinoma (OSCC) (Li et al. 4). The major advantage of this technique is that the anticancer effect on OSCC is virtually accomplished by cellular proliferation in addition to cellular apoptosis (inhibition of tumor angiogenesis) (Li et al. 4).

Monoclonal antibodies in target therapies of breast cancer

Globally, breast cancer is known to be a killer disease that largely affects females; the major causative agent in about 10% of world breast cancer cases is the mutation of the gene, which is inherited from any of the parents (Grammatikakis, Zervoudis & Kassanos 640). Furthermore, the most effective known therapeutic alternative diagnosis or treatment of breast cancer globally is the use of gene therapy. Some of the procedures used in treatment include molecular chemotherapy, antiangiogenic gene therapy, among many other therapies currently being used (Grammatikakis et al. 640).

bacteria-mediated anti-angiogenesis therapy

Most of the recent studies have revealed that there are some bacterial species that are capable of colonizing solid tumors. This inherited characteristic, however, can further be enhanced via genetic engineering, developing a natural anti-tumor activity that always enables the specific bacteria to transfer its therapeutic molecules directly into the target cells (Gardlik, Behuliak, Palffy, Celec & Li 7). There are few completed studies that have completely documented the anti-angiogenesis process, which is basically a bacterial mediated therapy for cancer (Gardlik et al. 7).

There are four recognized approaches that scientists use when utilizing the use of bacteria in cancer treatments; these include anticancer therapeutic-autofiction, DNA vaccination, transkingdom RNA interference, and alternative gene therapy (Gardlik et al. 7). Notable to mention is that the major primary goal of all these approaches is that they all focus on stimulation of angiogenesis suppression.

Based on the evidence of this paper, the discussion focuses on the argument of whether gene therapy is effective when the cost-benefit analysis is undertaken. Personally, I totally agree with the essay topic that gene therapy has more benefits than costs since it has been successful in the treatment of most chronic cancerous diseases. Gene therapy usually works by relying on immunotherapy and the use of vector organisms like viral particles to modify the genome of the host cell that triggers an immune response, which finally destroys the cancer cells present in the body (Cross & Burmester).

By using gene therapy, many cancerous diseases, i.e., lung, prostate, pancreatic cancers, have a higher chance of being completely treated. As such, gene therapy is now emerging to be the most common preferred treatment of choice because of its efficacy over other treatment methods (Cross & Burmester). The gene treatment also allows the use of a single vector or a combination of several vectors aimed at achieving optimal results (Cross & Burmester).

Currently, further research on gene therapy is still ongoing, and various clinical trials have so far been completed, which now has led to the evolution of vaccine productions (Cross & Burmester). The vaccines are promising to be the most effective technique since they only require autologous cells to have them manufactured, although this has many cost implications; this is one of the disadvantages of this technique. The second disadvantage is that very few hospitals globally are capable of having such vaccines manufactured because of the costly technology associated with the production of the vaccine. All these factors ultimately limit the availability of the vaccine as a viable treatment option.

A recent research study provides findings that show evidence of secondary gliomas stem cell, which originates from an astrocytic tumor that contains a genetic mutation that possesses the tumor suppressor gene recognized as p53 (Cross & Burmester). As part of the procedure, it was proposed in the experiments that in order to induce apoptosis of tumor cells, one must incorporate the use of an integrated suicide factor together with the adenovirus-mediated transfer of p53 (Cross & Burmester). Considerably, the main strength of this approach is supported by the fact that p53 mediate apoptosis follows two distinct and separate pathways reducing pathogenicity (Cross & Burmester).

Gene therapy technology has brought a great 21st-century revolution to the modern system of disease treatments, precisely when it comes to cancer treatments. It is remarkable to note that the development of anticancer treatments by modified immunotherapy and gene therapy, among others, has helped to cure many cases of cancers and saved many others from death. However, through gene therapy, many victims of cancer have attained a prolonged lifespan after being subjected to this treatment.

Scientifically, the principle behind gene therapy when it comes to cancer treatment is that a successful cancer treatment involving therapeutic modality always aims at real activation of death pathways within the cancer cells.

Therefore, there is no doubt that for cancerous diseases, the development of genetic engineering and specific cancer vaccines are proving to be the most effective treatment approaches. There are hope and confidence that in the near future, all obstacles encountered during the first generation cancerous and precancerous treatments will be eliminated by the development of second-generation modern therapeutic intervention as far as disease (cancer) treatments is concerned (Cross & Burmester).

Cross, Deanna., & James, Burmester. “ Gene Therapy for Cancer Treatment: Past, Present and Future .” Clinical Medicine and Research . 2006. Web.

Gardlik, R., Behuliak, M., Palffy, R., Celec, P., & Li, C. “Gene therapy for cancer: bacteria- mediated anti-angiogenesis therapy.” Clinigene Current Gene Therapy Weekly (2011): 7.

Grammatikakis, I., Zervoudis, S., & Kassanos, D. “Synopsis of new antiangiogenetic factors, mutation compensation agents, and monoclonal antibodies in target therapies of breast cancer.” Clinigene Current Gene Therapy Weekly (2011): 7.

Li, Y., Li, M., Yao, G., Geng, N., Xie, Y., Feng, Y., Zhang, P., et al. “Telomerase inhibition strategies by siRNAs against either hTR or hTERT in oral squamous cell carcinoma.” Clinigene Current Gene Therapy Weekly (2011): 4.

Rothchild, Allisa., Laura, Martin., & Lauren, Lubrano. “Gene Therapy and the Gametes.” Somatic Gene Therapy . 2010. Web.

Walesgenepark.co.uk . “What is Germline Gene Therapy.” Wales Gene Park . 2011. Web.

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• INNOVATIONS IN
• 26 October 2021

Four Success Stories in Gene Therapy

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After numerous setbacks at the turn of the century, gene therapy is treating diseases ranging from neuromuscular disorders to cancer to blindness. The success is often qualified, however. Some of these therapies have proved effective at alleviating disease but come with a high price tag and other accessibility issues: Even when people know that a protocol exists for their disease and even if they can afford it or have an insurance company that will cover the cost—which can range from $400,000 to $2 million—they may not be able to travel to the few academic centers that offer it. Other therapies alleviate symptoms but don’t eliminate the underlying cause.

“Completely curing patients is obviously going to be a huge success, but it’s not [yet] an achievable aim in a lot of situations,” says Julie Crudele, a neurologist and gene therapy researcher at the University of Washington. Still, even limited advances pave the way for ongoing progress, she adds, pointing to research in her patients who have Duchenne muscular dystrophy: “In most of these clinical trials, we learn important things.”

Thanks to that new knowledge and steadfast investigations, gene therapy researchers can now point to a growing list of successful gene therapies. Here are four of the most promising.

Gene Swaps to Prevent Vision Loss

Some babies are born with severe vision loss caused by retinal diseases that once led inevitably to total blindness. Today some of them can benefit from a gene therapy created by wife-and-husband team Jean Bennett and Albert Maguire, who are now ophthalmologists at the University of Pennsylvania.

When the pair first began researching retinal disease in 1991, none of the genes now known to cause vision loss and blindness had been identified. In 1993 researchers identified one potential target gene, RPE65 . Seven years later Bennett and Maguire tested a therapy targeting that gene in three dogs with severe vision loss—it restored vision for all three.

Part of Innovations In Gene Therapy

In humans, the inherited condition that best corresponds with the dogs’ vision loss is Leber congenital amaurosis (LCA). LCA prevents the retina, a layer of light-sensitive cells at the back of the eye, from properly reacting or sending signals to the brain when a photon strikes it. The condition can cause uncontrolled shaking of the eye (nystagmus), prevents pupils from responding to light and typically results in total blindness by age 40. Researchers have linked the disease to mutations or deletions in any one of 27 genes associated with retinal development and function. Until gene therapy, there was no cure.

Mutations in RPE65 are just one cause of inherited retinal dystrophy, but it was a cause that Bennett and Maguire could act on. The researchers used a harmless adeno-associated virus (AAV), which they programmed to find retinal cells and insert a healthy version of the gene, and injected it into a patient’s eye directly underneath the retina. In 2017, after a series of clinical trials, the Food and Drug Administration approved voretigene neparvovec-rzyl (marketed as Luxturna) for the treatment of any heritable retinal dystrophy caused by the mutated RPE65 gene, including LCA type 2 and retinitis pigmentosa, another congenital eye disease that affects photoreceptors in the retina. Luxturna was the first FDA-approved in vivo gene therapy, which is delivered to target cells inside the body (previously approved ex vivo therapies deliver the genetic material to target cells in samples collected from the body, which are then reinjected).

Spark Therapeutics, the company that makes Luxturna, estimates that about 6,000 people worldwide and between 1,000 and 2,000 in the U.S. may be eligible for its treatment—few enough that Luxturna was granted “orphan drug” status, a designation that the FDA uses to incentivize development of treatments for rare diseases. That wasn’t enough to bring the cost down. The therapy is priced at about $425,000 per injection, or nearly $1 million for both eyes. Despite the cost, Maguire says, “I have not yet seen anybody in the U.S. who hasn’t gotten access based on inability to pay.”

Those treated show significant improvement: Patients who were once unable to see clearly had their vision restored, often very quickly. Some reported that, after the injections, they could see stars for the first time.

While it is unclear how long the effects will last, follow-up data published in 2017 showed that all 20 patients treated with Luxturna in a phase 3 trial had retained their improved vision three years later. Bennett says five-year follow-up with 29 patients, which is currently undergoing peer review, showed similarly successful results. “These people can now do things they never could have dreamed of doing, and they’re more independent and enjoying life.”

Training the Immune System to Fight Cancer

Gene therapy has made inroads against cancer, too. An approach known as chimeric antigen receptor (CAR) T cell therapy works by programming a patient’s immune cells to recognize and target cells with cancerous mutations. Steven Rosenberg, chief of surgery at the National Cancer Institute, helped to develop the therapy and published the first successful results in a 2010 study for the treatment of lymphoma.

“That patient had massive amounts of disease in his chest and his belly, and he underwent a complete regression,” Rosenberg says—a regression that has now lasted 11 years and counting.

CAR T cell therapy takes advantage of white blood cells, called T cells, that serve as the first line of defense against pathogens. The approach uses a patient’s own T cells, which are removed and genetically altered so they can build receptors specific to cancer cells. Once infused back into the patient, the modified T cells, which now have the ability to recognize and attack cancerous cells, reproduce and remain on alert for future encounters.

In 2016 researchers at the University of Pennsylvania reported results from a CAR T cell treatment, called tisagenlecleucel, for acute lymphoblastic leukemia (ALL), one of the most common childhood cancers. In patients with ALL, mutations in the DNA of bone marrow cells cause them to produce massive quantities of lymphoblasts, or undeveloped white blood cells, which accumulate in the bloodstream. The disease progresses rapidly: adults face a low likelihood of cure, and fewer than half survive more than five years after diagnosis.

When directed against ALL, CAR T cells are ruthlessly efficient—a single modified T cell can kill as many as 100,000 lymphoblasts. In the University of Pennsylvania study, 29 out of 52 ALL patients treated with tisagenlecleucel went into sustained remission. Based on that study’s results, the FDA approved the therapy (produced by Novartis as Kymriah) for treating ALL, and the following year the agency approved it for use against diffuse large B cell lymphoma. The one-time procedure costs upward of $475,000.

CAR T cell therapy is not without risk. It can cause severe side effects, including cytokine release syndrome (CRS), a dangerous inflammatory response that ranges from mild flulike symptoms in less severe cases to multiorgan failure and even death. CRS isn’t specific to CAR T therapy: Researchers first observed it in the 1990s as a side effect of antibody therapies used in organ transplants. Today, with a combination of newer drugs and vigilance, doctors better understand how far they can push treatment without triggering CRS. Rosenberg says that “we know how to deal with side effects as soon as they occur, and serious illness and death from cytokine release syndrome have dropped drastically from the earliest days.”

Through 2020, the remission rate among ALL patients treated with Kymriah was about 85 percent. More than half had no relapses after a year. Novartis plans to track outcomes of all patients who received the therapy for 15 years to better understand how long it remains effective.

Precision Editing for Blood Disorders

One new arrival to the gene therapy scene is being watched particularly closely: in vivo gene editing using a system called CRISPR, which has become one of the most promising gene therapies since Jennifer Doudna and Emmanuelle Charpentier discovered it in 2012—a feat for which they shared the 2020 Nobel Prize in Chemistry. The first results from a small clinical trial aimed at treating sickle cell disease and a closely related disorder, called beta thalassemia, were published this past June.

Sickle cell disease affects millions of people worldwide and causes the production of crescent-shaped red blood cells that are stickier and more rigid than healthy cells, which can lead to anemia and life-threatening health crises. Beta thalassemia, which affects millions more, occurs when a different mutation causes someone’s body to produce less hemoglobin, the iron-rich protein that allows red blood cells to carry oxygen. Bone marrow transplants may offer a cure for those who can find matching donors, but otherwise treatments for both consist primarily of blood transfusions and medications to treat associated complications.

Both sickle cell disease and beta thalassemia are caused by heritable, single-gene mutations, making them good candidates for gene-editing therapy. The method, CRISPR-Cas9, uses DNA sequences from bacteria (clustered regularly interspaced short palindromic repeats, or CRISPR) and a CRISPR-associated enzyme (Cas for short) to edit the patient’s genome. The CRISPR sequences are transcribed onto RNA that locates and identifies DNA sequences to blame for a particular condition. When packaged together with Cas9, transcribed RNA locates the target sequence, and Cas9 snips it out of the DNA, thereby repairing or deactivating the problematic gene.

At a conference this past June, Vertex Pharmaceuticals and CRISPR Therapeutics announced unpublished results from a clinical trial of beta thalassemia and sickle cell patients treated with CTX001, a CRISPR-Cas9-based therapy. In both cases, the therapy does not shut off a target gene but instead delivers a gene that boosts production of healthy fetal hemoglobin—a gene normally turned off shortly after birth. Fifteen people with beta thalassemia were treated with CTX001; after three months or more, all 15 showed rapidly improved hemoglobin levels and no longer required blood transfusions. Seven people with severe sickle cell disease received the same treatment, all of whom showed increased levels of hemoglobin and reported at least three months without severe pain. More than a year later those improvements persisted in five subjects with beta thalassemia and two with sickle cell. The trial is ongoing, and patients are still being enrolled. A Vertex spokesperson says it hopes to enroll 45 patients in all and file for U.S. approval as early as 2022.

Derailing a Potentially Lethal Illness

Spinal muscular atrophy (SMA) is a neurodegenerative disease in which motor neurons—the nerves that control muscle movement and that connect the spinal cord to muscles and organs—degrade, malfunction and die. It is typically diagnosed in infants and toddlers. The underlying cause is a genetic mutation that inhibits production of a protein involved in building and maintaining those motor neurons.

The four types of SMA are ranked by severity and related to how much motor neuron protein a person’s cells can still produce. In the most severe or type I cases, even the most basic functions, such as breathing, sitting and swallowing, prove extremely challenging. Infants diagnosed with type I SMA have historically had a 90 percent mortality rate by one year.

Adrian Krainer, a biochemist at Cold Spring Harbor Laboratory, first grew interested in SMA when he attended a National Institutes of Health workshop in 1999. At the time, Krainer was investigating how RNA mutations cause cancer and genetic diseases when they disrupt a process called splicing, and researchers suspected that a defect in the process might be at the root of SMA. When RNA is transcribed from the DNA template, it needs to be edited or “spliced” into messenger RNA (mRNA) before it can guide protein production. During that editing process, some sequences are cut out (introns), and those that remain (exons) are strung together.

Krainer realized that there were similarities between the defects associated with SMA and one of the mechanisms he had been studying—namely, a mistake that occurs when an important exon is inadvertently lost during RNA splicing. People with SMA were missing one of these crucial gene sequences, called SMN1 .

“If we could figure out why this exon was being skipped and if we could find a solution for that, then presumably this could help all the [SMA] patients,” Krainer says. The solution he and his colleagues hit on, antisense therapy, employs single strands of synthetic nucleotides to deliver genetic instructions directly to cells in the body . In SMA’s case, the instructions induce a different motor neuron gene, SMN2 , which normally produces small amounts of the missing motor neuron protein, to produce much more of it and effectively fill in for SMN1 . The first clinical trial to test the approach began in 2010, and by 2016 the FDA approved nusinersen (marketed as Spinraza). Because the therapy does not incorporate itself into the genome, it must be administered every four months to maintain protein production. And it is staggeringly expensive: a single Spinraza treatment costs as much as $750,000 in the first year and $375,000 annually thereafter.

Since 2016, more than 10,000 people have been treated with it worldwide. Although Spinraza can’t restore completely normal motor function (a single motor neuron gene just can’t produce enough protein for that), it can help children with any of the four types of SMA live longer and more active lives. In many cases, Spinraza has improved patients’ motor function, allowing even those with more severe cases to breathe, swallow and sit upright on their own. “The most striking results are in patients who are being treated very shortly after birth, when they have a genetic diagnosis through newborn screening,” Krainer says. “Then, you can actually prevent the onset of the disease—for several years and hopefully forever.”

doi: https://doi.org/10.1038/d41586-021-02737-7

This article is part of Innovations In Gene Therapy , an editorially independent supplement produced with the financial support of third parties. About this content .

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Gene therapy: Comprehensive overview and therapeutic applications

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• 1 Department of Pharmacy, Pravara Rural Education Society's (P.R.E.S.'s) College of Pharmacy, Shreemati Nathibai Damodar Thackersey (SNDT) Women's University, Nashik 400020, Maharashtra, India.
• 2 Department of Biosciences and Bioengineering, Indian Institute of Technology (IIT), Roorkee, Roorkee, Uttarakhand 247667, India.
• 3 Centre for Biomedical Engineering (CBME), Indian Institute of Technology (IIT) Delhi, Hauz Khas, New Delhi 110016, India; Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Rajendranagar, Hyderabad 500030, Telangana, India; Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Mamnoor, Warangal 506166, Telangana, India; Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH Aachen University Hospital, Pauwelsstr. 30, D-52074 Aachen, Germany. Electronic address: [email protected].
• 4 Department of Pharmacology, Central University of Punjab, Ghudda, Bathinda 151401, Punjab, India.
• 5 The Business Research Company (TBRC), Jubilee Hills, Hyderabad 500033, Telangana, India.
• 6 Department of Pharmaceutical Chemistry, University Institute of Pharmaceutical Sciences (UIPS), Panjab University, Chandigarh 160014, India.
• 7 Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Rajendranagar, Hyderabad 500030, Telangana, India.
• 8 Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH Aachen University Hospital, Pauwelsstr. 30, D-52074 Aachen, Germany. Electronic address: [email protected].
• 9 Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Mamnoor, Warangal 506166, Telangana, India. Electronic address: [email protected].
• PMID: 35123997
• DOI: 10.1016/j.lfs.2022.120375

Gene therapy is the product of man's quest to eliminate diseases. Gene therapy has three facets namely, gene silencing using siRNA, shRNA and miRNA, gene replacement where the desired gene in the form of plasmids and viral vectors, are directly administered and finally gene editing based therapy where mutations are modified using specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short tandem repeats (CRISPR)/CRISPR-associated protein (Cas)-associated nucleases. Transfer of gene is either through transformation where under specific conditions the gene is directly taken up by the bacterial cells, transduction where a bacteriophage is used to transfer the genetic material and lastly transfection that involves forceful delivery of gene using either viral or non-viral vectors. The non-viral transfection methods are subdivided into physical, chemical and biological. The physical methods include electroporation, biolistic, microinjection, laser, elevated temperature, ultrasound and hydrodynamic gene transfer. The chemical methods utilize calcium- phosphate, DAE-dextran, liposomes and nanoparticles for transfection. The biological methods are increasingly using viruses for gene transfer, these viruses could either integrate within the genome of the host cell conferring a stable gene expression, whereas few other non-integrating viruses are episomal and their expression is diluted proportional to the cell division. So far, gene therapy has been wielded in a plethora of diseases. However, coherent and innocuous delivery of genes is among the major hurdles in the use of this promising therapy. Hence this review aims to highlight the current options available for gene transfer along with the advantages and limitations of every method.

Keywords: Gene delivery; Gene therapy; Non-viral vectors; Transfection; Viral vectors.

Copyright © 2022. Published by Elsevier Inc.

Publication types

• CRISPR-Cas Systems*
• Gene Editing*
• Gene Transfer Techniques*
• Genetic Diseases, Inborn / genetics
• Genetic Diseases, Inborn / therapy*
• Genetic Therapy*
• Genetic Vectors / therapeutic use*

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Guest Essay

The Future of Medicine Is Unfolding Before Us. Are We Nurturing It?

By Elizabeth Currid-Halkett

Dr. Currid-Halkett is a Guggenheim fellow and professor of public policy at the University of Southern California.

On Jan. 8, 2020, as I was parking my car, I got a long-awaited phone call from one of my son’s doctors. She informed me that our 7-month-old son, Eliot, had Duchenne muscular dystrophy, a fatal neuromuscular disease.

I can still remember the way the Los Angeles winter sunlight hit the dashboard. I can see my neighbor walking up her steps with groceries, a leaf falling, oblivious to the devastation below. “Life changes in an instant,” Joan Didion wrote. “The ordinary instant.” Our son had a fatal illness. He would die before us.

D.M.D. prevents the production of dystrophin, a protein needed to protect and repair muscle cells. It is caused by a genetic mutation on the X chromosome, thus the disease almost exclusively affects boys (one in 3,300). Over time, children with D.M.D. lose muscle mass and thus the ability to do basic things like run and walk. Eventually they lose their ability to breathe, and they experience heart failure. There is no known cure. While existing treatments have helped extend the life span of sufferers, they mainly focus on managing symptoms.

In my search for answers for how to save my son, I contacted Dr. Jerry Mendell, a now-retired neurologist at Nationwide Children’s Hospital in Columbus, Ohio, who was running clinical trials for an experimental gene therapy he developed to enable dystrophin production in boys with D.M.D. The treatment, now known as Elevidys, offered the prospect of not merely managing symptoms, but slowing the disease’s progression or even stopping it in its tracks — and potentially, for the first time in the history of this terrible disease, allowing boys with D.M.D. a chance to thrive.

Since I had that first conversation with Dr. Mendell (also a senior adviser for Sarepta, the maker of Elevidys), clinical trials for the gene therapy have had their ups and downs , and some adverse effects have been reported. But in June 2023, based on a two-part clinical trial, the Food and Drug Administration granted accelerated approval for the treatment for 4- and 5-year-olds who do not have other disqualifying conditions. The F.D.A.’s approval was contingent on continuing trials showing evidence of improved motor function, which had not yet been established.

Before Eliot received his treatment, he had difficulty going up stairs. He complained about being tired after walking only a block or two, even on Halloween, when candy ought to have motivated him. Hopping on one foot, a milestone for a 4-year-old, was impossible.

On Aug. 29, he finally received the one-time infusion. Three weeks later, he was marching upstairs and able to jump over and over. After four weeks, he could hop on one foot. Six weeks after treatment, Eliot’s neurologist decided to re-administer the North Star Ambulatory Assessment , used to test boys with D.M.D. on skills like balance, jumping and getting up from the floor unassisted. In June, Eliot’s score was 22 out of 34. In the second week of October, it was a perfect 34 — that of a typically developing , healthy 4-year-old boy. Head in my hands, I wept with joy. This was science at its very best, close to a miracle.

But the goal to offer this possible future to more patients with D.M.D. is in jeopardy. Sarepta is seeking F.D.A. approval to treat boys over 5 . Disagreements over the latest clinical trial’s results threaten to derail that outcome.

Moreover, what the F.D.A. decides to do next with Elevidys could set the tone for how it handles other emerging gene therapies for rare diseases. We can already see roadblocks that prevent more families from gaining access to these new treatments — from high costs and insurance challenges to dissent over how flexible regulators should be in interpreting clinical trial results and taking qualitative improvements into account. What is at stake with the debate around Elevidys is more than just the chance to give other boys with D.M.D. a more normal life. The challenges that we are witnessing with Elevidys are a harbinger of the fights we may see with gene therapies developed for other rare diseases.

There’s an opportunity to reduce those barriers now, while these treatments are still in their early phases. Every child afflicted with a life-threatening disease deserves the chance Eliot has been given.

The biggest obstacle to getting these treatments is cost. Gene therapies cost, on average, $1 million to $2 million . At $3.2 million per patient, Elevidys is the second-most-expensive drug in the world . Insurance companies would probably prefer not to foot the bill, and without full F.D.A. approval, insurance companies can refuse to cover these treatments by claiming they are medically unnecessary or experimental . Before Eliot’s treatment began, my insurance company initially said it would cover the cost but then started stalling on coverage and questioning the urgency of Eliot’s treatment. I was able to call Dana Goldman, the dean of the Sol Price School of Public Policy at the University of Southern California, where I work, to help me navigate the process. I was in the rare position to marshal resources and assistance to pressure my insurance company into covering Elevidys. Across the country, physicians are fighting denials and seeking appeals for their young patients.

Dr. Goldman has argued that one way to incentivize insurance companies to cover the high costs of treatments like gene therapies is to amortize how much the companies pay over time if the effectiveness of such treatments does not last (analogous to a pay-for-performance model). Another option is for pharmaceutical companies to offer a warranty that gives a prorated refund to the insurance company if a patient needs to return to prophylaxis treatment within a certain number of years. Costs are an especially frustrating problem for rare diseases like D.M.D., for which the extremely small patient population deters companies from investing money and resources to develop new treatments. Some experts believe the federal government ought to do more to directly complement research funding for rare diseases , as it has through the Orphan Drug Act for over four decades. The government could also defray the cost to consumers by offering subsidies directly to patients.

There’s another big role the government can play to accelerate gene therapies besides intervening in costs, and that’s to make the wheels of regulatory approval for these drugs less onerous. Flexibility doesn’t have to come at the cost of safety. The F.D.A. acted swiftly to approve an antiretroviral drug for H.I.V. in the 1980s and the Covid vaccines in December 2020, saving millions of lives without putting people in harm’s way.

But Elevidys is a case study in how the F.D.A. can get in its own way. D.M.D. patients 4 or 5 years old received access to the drug under fast-tracked approval, the first time a drug was approved under this new framework. But this was reportedly only because Peter Marks , the director of the F.D.A.’s Center for Biologics Evaluation and Research, disagreed with his own staff’s rejection . Current concern over Elevidys’s approval for boys over 5 focuses on the most recent clinical trial results , which showed older boys, whose muscular decline is further along, did not improve on motor function as measured by the North Star Ambulatory Assessment after treatment. However, as Sarepta has noted, they still saw gains in their ability to rise from the floor and walk 10 meters, indicating possible slowing of the disease that could significantly improve and extend their lives.

Detractors suggest this improvement is not enough to meet the bar for approval. This is a common problem for rare disease trials because they often consist of very few participants. In such cases, a narrow focus on numbers ignores the real quality-of-life benefits doctors, patients and their families see from these treatments. During the advisory committee meeting for Elevidys in May 2023, I listened to F.D.A. analysts express skepticism about the drug after they watched videos of boys treated with Elevidys swimming and riding bikes. These experts — given the highest responsibility to evaluate treatments on behalf of others’ lives — seemed unable to see the forest for the trees as they focused on statistics versus real-life examples.

The F.D.A. can have a more flexible view of treatment efficacy without losing focus on safety. As with any drug, whether for migraines or asthma, there will be a spectrum of effectiveness. The same will be true of all gene therapies, and the F.D.A. should reconsider the metrics it uses to green-light these treatments now, before it potentially leaves thousands of patients in the lurch, out of access to something lifesaving.

Gene therapy is the future of medicine. Our bureaucracy and insurance companies should not hinder patients from receiving pioneering treatments that could transform their lives. As parents, we are not asking for the moon. We just want our children to live.

Elizabeth Currid-Halkett is a Guggenheim fellow and professor of public policy at the University of Southern California.

The Times is committed to publishing a diversity of letters to the editor. We’d like to hear what you think about this or any of our articles. Here are some tips . And here’s our email: [email protected] .

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• Open access
• Published: 24 May 2024

Molecular mechanisms of aging and anti-aging strategies

• Yumeng Li 1 ,
• Xutong Tian 1 ,
• Juyue Luo 1 ,
• Tongtong Bao 1 ,
• Shujin Wang 2 &

Cell Communication and Signaling volume  22 , Article number:  285 ( 2024 ) Cite this article

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Aging is a complex and multifaceted process involving a variety of interrelated molecular mechanisms and cellular systems. Phenotypically, the biological aging process is accompanied by a gradual loss of cellular function and the systemic deterioration of multiple tissues, resulting in susceptibility to aging-related diseases. Emerging evidence suggests that aging is closely associated with telomere attrition, DNA damage, mitochondrial dysfunction, loss of nicotinamide adenine dinucleotide levels, impaired macro-autophagy, stem cell exhaustion, inflammation, loss of protein balance, deregulated nutrient sensing, altered intercellular communication, and dysbiosis. These age-related changes may be alleviated by intervention strategies, such as calorie restriction, improved sleep quality, enhanced physical activity, and targeted longevity genes. In this review, we summarise the key historical progress in the exploration of important causes of aging and anti-aging strategies in recent decades, which provides a basis for further understanding of the reversibility of aging phenotypes, the application prospect of synthetic biotechnology in anti-aging therapy is also prospected.

Aging will be a major social problems worldwide in the coming decades [ 1 , 2 , 3 ]. During the aging process, the body tissues and organs of the older people undergo functional decline or deterioration, thus increasing their susceptibility to age-related diseases and shortening their healthy life span, which has brought enormous financial pressure to countries worldwide in terms of pension, medical expenses, and health care [ 4 , 5 , 6 ]. Therefore, exploring the biological nature of aging, searching for safe and effective intervention strategies to positively regulate health status, and prolonging the healthy lifespan of the aging population are important for reducing the global pension burden and promoting healthy aging.

Aging is a progressive degenerative state that can be physiological and pathological [ 7 , 8 , 9 ] (Fig.  1 ). Physiological aging is observed in across many species, and is a degenerative process that occurs after maturation, including telomere attrition [ 10 , 11 ], DNA damage [ 12 , 13 ], mitochondrial dysfunction [ 14 , 15 ], loss of nicotinamide adenine dinucleotide (NAD + ) levels [ 16 , 17 ], impaired macro-autophagy [ 18 , 19 ], stem cell exhaustion, inflammation [ 20 , 21 ], loss of protein balance [ 22 ], deregulated nutrient-sensing [ 23 ], altered intercellular communication [ 24 , 25 , 26 ] and dysbiosis [ 27 , 28 ], thereby leading to systemic functional decline. Importantly, these changes are decentralised and interactive, not independent of each other. Pathological aging includes the senile pathological aging changes, which are caused by various external factors, such as cardiovascular disease [ 29 ], cerebrovascular disease [ 30 ], degenerative joint disease [ 31 , 32 ], diabetes [ 33 ], Parkinson’s disease [ 34 , 35 ], Alzheimer’s disease [ 36 ], cancer [ 37 , 38 , 39 ], and degeneration of multiple organ functions. These aging-induced cellular physiological and pathological changes can reflect the underlying nutrient sensing, intercellular communication, protein stabilisation, epigenetics, and molecular abnormalities in DNA damage repair, leading to genomic instability and damage. Further understanding of the different molecular mechanisms involved in the aging process is of great importance for preventing aging and prolonging the lifespan.

Aging drivers and age-related diseases. Major physiological features of aging include NAD + loss, telomeres attrition, mitochondrial dysfunction, stem cell exhaustion, disabled macro-autophagy, DNA damage, protein balance loss, inflammation, dysbiosis, deregulated nutrient sensing, and altered cellular communication. These physiological characteristics of aging are primitive, antagonistic, and integrated, and their interaction promotes aging. When aging reaches a certain threshold, organ and tissue function continues to deteriorate, which increases the incidence and mortality of aging-related diseases, including cardiovascular, cerebrovascular, degenerative joint disease, diabetes, Parkinson’s disease, Alzheimer’s disease, and cancer

In recent years, a large number of animal and clinical experiments have been conducted to study factors that induce aging, such as morphological and pathological changes and functional decline of the aging organism. Indeed, some differences between biological and chronological age reflect the validity of age-accelerated or deceleration procedures, which are well-known biomarkers of the aging process. Researchers have gradually expanded from traditional methods of measuring aging (including maximal energy expenditure at the respiratory, sensory, psychomotor, and cognitive levels) to modern biotechnological methods, such as genomics, epigenomics, transcriptomics, proteomics, and metabolomics. These techniques may have implications for assessing the spatiotemporal patterns of health degradation and effectiveness of anti-aging strategies.

Briefly, aging is a complex process, and its characteristics are interdependent. Each of these factors should be considered as an entry point for future exploration of the aging process and the development of novel life extensions. Here, we review the history and current state of aging research and summarise the characteristics of aging and the mechanisms promoting aging. In addition, we review different types of aging mechanisms and their corresponding anti-aging strategies. This knowledge can guide the design of preventive and therapeutic strategies to delay aging and age-related diseases and extend human health and longevity.

Potential triggers and molecular mechanisms of aging

Aging is a complex result of many biological processes, and many key factors trigger aging, such as DNA damage, telomere dysregulation, mitochondrial dysregulation, NAD + loss, autophagy disorders, and stem cell exhaustion. Here, we summarise the main causes and underlying molecular mechanisms contributing to the aging process.

Aging and DNA damage

DNA damage is a major internal factor that leads to genomic instability, epigenetic changes, protein stress, impaired mitochondrial function, and telomere dysfunction [ 12 ]. The continuous accumulation of DNA-damaged cells triggers cell death and senescence, ultimately leading to chronic inflammation, loss of function, atrophy, and disease in cells and tissues [ 40 ].

Molecular mechanisms of DNA damage

Genomic instability manifests as permanent and transmissible changes in DNA sequence [ 13 , 41 ]. DNA damage caused by an inherently unstable genome includes spontaneous deamination, hydrolysis, and many other chemical changes such as different types of breaks, changes in base positions, gaps, DNA-protein cross-links, and other subtle chemical modifications [ 12 , 42 ]. Abnormal DNA structures (e.g. G-quadruplexes, R-loops, and persistent single-stranded regions), as well as abnormal intermediates in DNA transactions (e.g. stalled transcription, replication, and recombination complexes), are considered phenotypes of DNA damage [ 13 ]. Genomic mutations caused by DNA instability adversely affect cellular functions and are major causes of cancer and genetic diseases. However, DNA instability is also the most important substrate in the evolution of species [ 43 , 44 ]. DNA integrity is maintained by the continuous repair of highly complex DNA repair and DNA damage response (DDR) systems that counteract the time-dependent erosion and destruction of genetic DNA information. Progressive telomere shortening is another major contributor to DNA damage that accelerates the aging process [ 45 ].

DNA damage is the major driver of age-related epigenetic changes. The epigenome, which includes DNA methylation and histone modifications, is unstable throughout the life cycle of somatic cells [ 46 ]. DNA damage leads to persistent chromatin changes that enrich aging-enhancing DNA fragments (DNA-scars) in senescent cells [ 47 ]. Persistent DNA damage and repair-related cellular physiological effects may leave epigenetic marks, resulting in epigenetic heterogeneity among cells. Transcription appears to change considerably more in senescent cells than in young cells. Thus, DDR may be a major cause of epigenetic changes that impair gene expression control, leading to somatic heterogeneity and a time-dependent decline in overall function.

Relationship between DNA damage and aging

During aging, numerous exogenous and endogenous genotoxins, photoaging, and mechanical stress in tissues continuously induce DNA damage (Fig.  2 ). Approximately 10 5 DNA damage events occur in mammalian cells every day, although most of the DNA damage is effectively excised or repaired. Notably, a small portion escapes the DNA damage detection and repair system, subsequently resulting in failure to repair or repair errors [ 48 ]. Many studies using mammalian models have confirmed an inextricable link between DNA damage and aging [ 49 , 50 , 51 , 52 ]. As aging progresses, the DNA repair capacity gradually declines, and the increased molecular phenotype of genomic instability becomes the main marker of aging. Markers of DNA damage are found in patients with age-related diseases such as cardiovascular disease [ 53 ], Alzheimer’s disease [ 54 ], and cancer [ 55 ], suggesting that DNA damage is directly related to the incidence of these diseases. Patients with genetic or acquired defects in DNA repair proteins also exhibit features of premature aging and that differences in the location of the defect in the DNA repair system can lead to premature aging in different organs [ 56 ]. Specifically, RecQ helicase plays an important role in DNA recombination, replication, repair, and telomere maintenance, and its mutation may increase the incidence of Werner, Bloom, and Rothmund-Thomson syndromes [ 57 ]. Global genome nucleotide excision repair deficiency leads to a thousand-fold increase in skin cancer susceptibility and may accelerate neurodegeneration [ 58 ]. Impaired transcription-coupled repair mechanisms can cause typical age-related pathologies, such as neurodegeneration, osteoporosis, and atherosclerosis [ 59 ]. Hutchinson-Gilford progeria is associated with nuclear genome instability, defects in DNA double-strand break repair leading to telangiectasia and Nijmegen break syndrome, and defects in DNA cross-linking repair leading to anaemia [ 60 ]. In addition, DNA damage caused by mitochondrial defects is another underlying factor in a class of progressive diseases that affect multiple organs.

Drivers of DNA damage and the resulting systemic consequences. The nuclear and mitochondrial genomes are constantly exposed to exogenous substances (such as ultraviolet and X-rays, chemicals in food, water, and air), endogenous substances such as ROS, advanced glycation end products (AGEs), and aldehydes; this results in genetic abnormalities, including mutation, deletion, aneuploidy, translocation, dysfunctional telomeres, epigenetic alterations, and mitochondrial dysfunction. DNA damage and DNA damage response caused by the above factors can shock molecular processes and alter cell fate, such as cell death, senescence, and systemic breakdown of repair functions, eventually leading to the loss of cell and organ function and promoting the occurrence and development of age-related diseases

Overall, defects in the DNA damage repair system directly leads to the continued accumulation of genomic mutations, which underlie many segmental forms of premature aging in humans, suggesting a close link between genome integrity and aging. Although considerable progress has been achieved in the study of the mechanistic connection between DNA damage and aging, there are still many issues to be further explore the specific molecular mechanisms by which DNA damage affects diseases in older people. Therefore, fundamentally addressing the aging process and combating age-related diseases are important for exploring the relationship between DNA damage and anti-aging effects.

Aging and telomere attrition

Telomeres are small stretches of DNA-protein complexes present at the ends of linear chromosomes in eukaryotic cells, which maintain chromosomal integrity, control the cell division cycle, and are essential for an organism’s healthy life span and reproduction [ 61 ]. As early as the 1960s, a scientist named Leonardo Hayflick discovered that cultured human fibroblasts had limited and reproducible replication capacity and were governed by cell-autonomous mechanisms [ 62 , 63 ]. Even if the cold stops the cell division, once the temperature rises again, the cells will continue to divide before freezing, until 50 times after the cessation of division. Heverick realized that cells have a deep-seated internal mechanism that controls the number of times they divide [ 64 ]. In the 1970s, Olovnikov [ 65 ] and Watson [ 66 ] discovered the “end duplication problem” by looking at asymmetries in linear DNA replication and predicting that each cell division results in chromosomal DNA at the ends of the lagging strands loss, eventually leading to the gradual shortening of chromosomes. Limited telomere length reserve is an obstacle to cell proliferation and viability, and the loss of telomere function is closely associated with age-related functional decline and increased incidence of disease [ 67 ] (Fig.  3 A).

Telomere and telomerase structure, and their relationship with cell senescence. A  Telomeres shorten during cell division, leading to accumulation of senescent cells. B  The structure of the telomere-telomerase complex. TERT, telomerase reverse transcriptase; TERC, telomerase RNA component; NOP10, nucleolar protein family A, member 3; NHP2, nucleolar protein family A, member 2; GAR, nucleolar protein family A; TIN2, TERF1-interacting nuclear factor 2; TPP1, telomere protection protein 1; TRF1, telomeric repeat binding factor 1; TRF2, telomeric repeat binding factor 2; POT1, protection of telomeres 1; RAP1, TERF2-interacting protein. The telomere diagram is derived from “biorender”

Telomere and telomerase structure

Telomere end protection is evolutionarily highly conserved from lower to higher multicellular organisms [ 68 ]. Structurally, telomeres consist of repeating nucleotide sequences 3’-[TTAGGG]-5’ in tandem, ranging from a few to tens of bases, terminated at the 3’ end by a single strand of guanine-rich nucleotides of 75 to 300 nt, forming a “cap structure” (Fig.  3 B). Telomeres are covered by a special protein called the shelterin complex, which is a multimer of six protein subunits (TRF1, TRF2, TPP1, POT1, TIN2, and RAP1) that work together to protect the chromosomes and regulate telomere length [ 68 ]. Telomeres and shelterin complexes form a sophisticated higher-order structure that protects DNA repair programmes from fusing ends by mediating non-homologous end-joining of telomeric DNA through double-stranded DNA break detection, ultimately involved in the capping, protection, and regulation of telomeres [ 69 ]. Correspondingly, mutations in these six protein components can disrupt the shelterin-telomere complex, resulting in terminal fusion and premature senescence. Specifically, telomere maintenance is inseparable from normal expression of TRF1 [ 70 , 71 ]. TRF1 deletion induces telomeric DNA to form a fragile site phenotype, whereas TRF1 overexpression impairs telomerase binding to telomere ends, eventually resulting in telomere shortening [ 72 , 73 ]. TRF2 folds telomeric DNA into T-loops, inhibits the ataxia telangiectasia mutated-dependent DDR at chromosome ends, and suppresses end-to-end chromosome fusion and canonical homologous end joining [ 74 ]. In addition, TIN2 plays a connecting role in the shelterin complex and forms bridges between different shelterin proteins [ 75 ]. TIN2 mutations do not interfere with the spatial structure of other shelterin components on telomeres; however, the TIN2-R282H mutation activates telomeric DNA damage signalling, which results in telomere instability associated with telomerase activity, eventually leading to a premature cellular senescence phenotype [ 76 ]. Uncontrolled POT1 impairs telomerase binding to telomere ends, resulting in shortened telomeres [ 77 ]. TPP1 interacts with telomerase reverse transcriptase (TERT) to recruit telomerase and its loss elicits a robust telomeric DNA damage response [ 78 ]. Rap1 is a key telomere-capping protein that prevents non-homologous end joining and telomere fusion, and its overexpression causes histone loss and accelerates cellular senescence [ 79 , 80 ]. Overall, the biological functional integrity of telomeres depends on the interaction of telomeres and the shelterin complex, which together regulate telomere length and the cell life cycle. It should be noted that normally shortened telomeres alone do not drive senescence (biology) if telomeres become so short that they are perceived as double-stranded DNA breaks, then these telomeres will recruit the DDR and induce the cells into a normal apoptotic or senescence program.

Telomerase is a riboprotease composed of two basic subunits: TERT and telomerase RNA component (TERC) [ 81 ]. The H/ACA domain of Cajal body protein 1 in TERC binds to telomerase to form telomerase Cajal body protein 1, which catalyzes telomerase activity and transports telomerase to the ends of telomeres [ 82 ]. In addition, multiple core protein components, including dyskerin, NHP2, NOP10, and GAR1, are essential for the normal catalytic function of telomerase [ 83 ]. Normally, telomerase is abundantly expressed in undifferentiated stem [ 67 ] and progenitor cells of germ cells [ 84 ], the skin, intestine [ 85 ], haematopoietic system [ 82 ], hair bulge [ 86 ], and testis [ 7 ]. Nevertheless, it is extremely low or undetectable in differentiated adult cells, such as neuroblasts [ 87 ], fibroblasts [ 88 ], cardiomyocytes [ 89 ], and sperm cells [ 90 ]. In the germ line and in some stem cells, telomerase can compensate for this loss of telomere duplication, which decreases with cell division [ 91 ]. Telomerase is silent during the early development of most somatic cells, limiting the number of cell divisions until the telomeres become very short [ 92 ]. The pathogenicity of telomere shortening during aging is a characteristic antagonistic pleiotropic effect. On the one hand, cells with telomere dysfunction are prone to genome instability and may become cancer cells. On the other hand, the normal replicative shortening of telomeres can restrict unrestricted cell proliferation and induce cell apoptosis or senescence, thus preventing the formation of tumors. Robinson et al. found a way to help telomeres maintain their length, a technique known as alternative lengthening of telomeres (ALT) [ 93 ]. In osteosarcoma and bread cancer cell lines, the potential relationship between telomere lengthening and inhibition of tumor growth is cleverly orchestrated in cell lines that maintain telomere length by the ALT [ 94 ]. It helps that tumors can be suppressed even when telomeres are lengthened.

Maintenance of adequate telomere length in normal cells requires intact telomere structure and highly sophisticated regulation of telomerase [ 95 ]. However, each associated protein in the telomere and telomerase complexes is susceptible to uncontrollable factors in the tissue microenvironment [ 96 ]. However, there is still some scientific debate regarding how the telomerase complex is sensed, expressed, and recruited to telomere ends for functional regulation to determine the role of telomeres and telomerases in the pathogenesis of systemic aging and degenerative diseases. Recently, telomere dysfunction has been described as a molecular feature of senescent cells, and the loss of telomere function is closely associated with genomic instability [ 97 ], DDR [ 98 ], and age-related decline in fitness [ 99 ]. Most importantly, telomere dysfunction during aging can amplify and drive other aging mechanisms and the progeria syndrome.

Relationship between telomere and telomerase dysfunction and aging

Organismal cellular telomere reserves are limited, and the loss of telomere function is closely associated with age-related adaptive decline [ 99 , 100 , 101 ] (Fig.  4 ). Excellent telomere and telomerase structures are essential for ensuring the normal physiological function of mothers and offspring, and their integrity has a certain genetic intergenerational effect [ 102 , 103 ]. Mice with knockout of TERT that are crossbred in successive generations, the telomeres of the offspring gradually shorten, finally developing telomere dysfunction in the third generation [ 104 ]. Additionally, low telomerase levels and continued tissue turnover lead to decades of progressive telomere attrition in the progenitor cells of highly proliferative tissues, including the haematopoietic system, gastrointestinal tract, and skin [ 10 , 11 ]. Excessive telomere attrition ultimately triggers DDR such as cell cycle arrest [ 105 ], apoptosis [ 106 , 107 ], differentiation disorders [ 108 ] and senescence [ 109 ]. Notably, as the aging process progresses, hypoproliferative tissues, including the heart, brain, and liver, may suffer from the effects of reactive oxygen species (ROS), which further induce telomere sequence damage, telomere attrition, and uncapping [ 86 , 110 ]. Thus, the aforementioned telomere properties make them a focal point in the biology of aging.

Telomere dysfunction activates DDR to drive cellular senescence. ROS induce telomere sequence damage, leading to telomere shortening and decapitation, triggering DDR, inducing the overexpression of cell cycle inhibition markers p53 and p21, and accelerating cell senescence. Senescent cells secrete SASP, which alter extracellular matrix composition, recruit and enhance T cells and macrophages, which can spread the aging phenotype to surrounding cells, thus promoting systemic chronic inflammation and inflammation-related diseases

Shortening of telomeres to a critical length leads to replicative cellular senescence [ 86 , 111 , 112 , 113 ]. Chromosomal telomeres gradually shorten as DNA replicates. When telomeres reach a critical length, they cannot bind enough telomere-covering proteins and are perceived as exposed DNA ends [ 114 ]. One or a few very short telomeres are sufficient to trigger the DNA damage response and induce overexpression of the cell cycle inhibitory markers p53 and p21, thereby forcibly inhibiting cell proliferation [ 115 ]. Accumulated senescent cells secrete a complex set of pro-inflammatory cytokines, termed the senescence-associated secretory phenotype (SASP), including interleukins, interleukin chemokines, proteases, and growth factors. The SASP alters the composition of the extracellular matrix and propagates the senescent phenotype to surrounding cells, leading to systemic chronic inflammation [ 116 ]. Interestingly, persistent telomere cohesion protected aged cells from premature senescence [ 117 ]. Therefore, telomere dysfunction-associated DNA damage response signalling events are key determinants of cell fate and organismal aging.

In summary, telomeres and telomerase play important roles in the core mechanisms that drive aging and many major human diseases. However, many knowledge gaps remain, such as the elucidation of the mechanisms regulating telomerase expression and activity, the non-canonical function of TERT, and the interactions between telomere dysfunction, inflammation, fibrosis, and degenerative disease. Therefore, there is an urgent need to develop telomerase activators for the treatment of aging and age-related diseases to prevent and treat fatal diseases caused by telomere shortening by rescuing telomeres and telomerase damage.

Aging and mitochondrial dysfunction

Mitochondria are the only organelles that retain their own genome and transcriptional and translational machinery, and are important cellular organelles for cellular energy conversion and signalling. The functional integrity of mitochondria is affected by intramitochondrial protein folding, mitochondrial membrane dynamics, mitosis, and intracellular environmental stress products. One of the classic features of aging is a progressive decline in mitochondrial activity and stress resilience. Mitochondrial dysfunction is closely associated with aging and age-related metabolic diseases.

Mitochondrial dysregulation by pleiotropic stress pathways

A healthy mitochondrial network generates adenosine triphosphate (ATP) through the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation, which maintain the basic energy conversion and information exchange within the cell and are essential for life [ 118 ]. Studies have shown that in normal cells, the nuclear gene-encoded transcription factor PCG1NRF1 induces the expression of mitochondrial-encoding genes, which further regulate mitochondrial biogenesis or increase mitochondrial activity to regulate cellular energy metabolism [ 119 ]. Conversely, metabolic perturbations of mitochondrial physiology, such as intramitochondrial protein stabilisation stress, energy deficit, and increased ROS production, trigger transcriptional reprogramming of nuclear genes for metabolic adaptation [ 120 ]. Notably, nuclear genes encode most of the mitochondrial proteome, whereas only a few protein-coding genes are encoded by the circular mtDNA. Therefore, to ensure protein balance and functional stability of the mitochondria, it is necessary to maintain excellent mitochondrial-nuclear genome-encoded communication channels [ 121 ].

In addition, mitochondria are the main cellular organelles that regulate energy homeostasis in cellular metabolism, and the dynamic balance of small molecules (including adenosine 5’-monophosphate (AMP), nicotinamide adenine dinucleotide (NAD + ), oxygen, ROS, and TCA cycle components) produced by mitochondria affect the information of mitochondria, nucleus, and other cellular organelles [ 14 ]. Specifically, ATP is a sensitive signal of mitochondrial health, and a continuous decrease in intracellular ATP levels increases the relative AMP content and activates the AMP-protein kinase signalling pathway [ 122 ]. The activated 5’-AMP-activated protein kinase (AMPK) signalling pathway further regulates key enzymes in other metabolic pathways (including fat and glucose metabolism, mitochondrial dynamics, autophagy, and protein synthesis) through phosphorylation and indirectly restores the energy balance in the mitochondria [ 123 , 124 ]. Disruption of this mechanism results in various mitochondria-related diseases. Similarly, NAD + is a cofactor for many metabolic reactions and a key factor in sensing the mitochondrial metabolic state and communicating it to other cellular organelles. We will elaborate on the important role of NAD + in the aging process in Sect.  2.4 . Oxygen is another small molecule that affects mitochondrial function; low intracellular oxygen levels reduce the ability of mitochondria to generate ATP [ 125 ]. Under normal conditions, cells can stabilise the structure of the proline hydroxylase domain of hypoxia-inducible factor-1/2a, limiting the potential impairment of mitochondrial function caused by low oxygen supply. In addition, toxic byproducts, such as ROS generated in mitochondria, can act on mitochondrial permeability pores together with excess Ca 2+ in mitochondria, resulting in oxidative damage and swelling of the mitochondria, thereby triggering inflammation and affecting mitochondrial function [ 126 ]. Small molecules in the TCA cycle, such as acetyl-CoA, α-ketoglutarate, succinic acid, and fumaric acid, are all signalling molecules that characterise the physiological state of mitochondria.

Relationship between mitochondrial dysfunction and aging

Mitochondrial dysfunction has pleiotropic effects (Fig.  5 ). Maintaining healthy and excellent mitochondrial metabolic function is a key factor in ensuring long-term health during the aging process, and the genetic stability of mtDNA and nuclear DNA determines the energy supply capacity of an organism’s tissues throughout life [ 121 ]. Unlike mitosis of the nuclear genome, mtDNA can replicate continuously, independently of the cell cycle. Owing to the low repair efficiency of the mtDNA repair system, mutated mtDNA copies accumulate in the cells over time. When the life cycle of an organism enters the later stages of life, heterogeneous mutations generated in both nuclear DNA and mtDNA exceed a certain threshold. These harmful physiological consequences promote the process of aging and age-related diseases, including disturbed glycolipid metabolism, reduced recognition knowledge, and shortened lifespan. Studies have reported that mtDNA mutant mice are more likely to develop signs of premature aging, such as a shortened lifespan, reduced fertility, anaemia, osteoporosis and hearing loss [ 127 ]. Notably, perturbation of the mtDNA epigenome has also been implicated in human progeria and disease [ 119 , 128 ]. The methylation of mtDNA is an important epigenetic modification. During the life cycle, mtDNA methylation is susceptible to environmental interference, endogenous metabolites, and other factors. Studies have found that individuals with reduced methylation in the D-loop region of mtDNA are more likely to develop amyotrophic lateral sclerosis and Parkinson’s disease, whereas those with reduced methylation of Mt-ND1 are more likely to develop Alzheimer’s disease [ 129 ] owing to the effect of mitochondrial dysfunction on normal cells. Conversely, senescent cells display changes in mitochondrial morphology, physiology, dynamics, and function. Studies have reported decreased mitochondrial membrane potential, increased proton leakage, and ROS production in senescent cells, further reducing cellular fatty acid oxidation and disrupting mitochondrial metabolism.

Mitochondrial dysfunction has pleiotropic effects in aging. Inducers such as the accumulation of mtDNA mutations, release of damaged toxic mitochondrial material, the production of mtROS, proteotoxicity, and deregulated metabolites (TCA intermediates, NAD + ) all contribute to mitochondrial dysfunction. Alterations in mitochondrial function have widespread adverse effects on intracellular homeostasis and lead to systemic organ decline and the development of several age-related diseases through complex signalling mechanisms (involving mitogens, metabolites, etc.)

In summary, many factors impair mitochondrial function during the life cycle, among which excessively reduced ATP, NAD + and oxygen levels, excessively accumulated ROS levels, and disrupted TCA cycle small molecules are the major contributors. Correspondingly, mitochondrial dysfunction is mainly reflected in transcriptional and epigenetic regulation caused by mitochondrial stress responses, such as mtDNA mutation, and the induction of other cellular organelle disorders, such as lysosomal storage disorders, impaired mitochondrial removal disorders, endoplasmic reticulum response, and changes in the cytoplasmic microenvironment. Based on the sensitivity of mitochondria to their microenvironment, mitochondrial dysfunction has been identified as an important trigger for aging and aging-related metabolic diseases. However, more research is needed to elucidate the interrelationships between mitochondrial dysfunction, aging, and aging-related diseases, as well as the underlying mechanisms of action, to discover new targets for anti-aging interventions.

Aging and NAD + loss

Nicotinamide adenine dinucleotide (NAD + ) is an important cofactor in the nucleus, cytoplasm, and mitochondria [ 130 ]. NAD + is involved in the regulation of cell redox reactions and energy metabolism, and its abnormal metabolism can affect cell metabolism, DNA repair, organelle function, immune cell viability, and cell aging [ 131 ]. However, aging is accompanied by a gradual decline in NAD + levels in tissues and cells, which accelerates the aging process and increases the prevalence of age-related diseases. Therefore, maintaining NAD + levels in tissue cells is important to alleviate the loss of tissue cell function, stabilise metabolic homeostasis, and promote healthy aging.

NAD + regulatory network and its role in cellular processes

NAD + is an important coenzyme in cellular redox reactions and is at the centre of energy metabolism [ 132 ]. It is involved in regulating the activity of dehydrogenase in metabolic pathways such as cellular glycolysis, fatty acid oxidation, and L-glutamine metabolism [ 133 ]. In these reactions, NAD + receives hydrogen ions, forms its reduced form NADH, transfers the accepted electrons to the electron transport chain, and generates ATP to supply energy to the cell. Conversely, NAD + is phosphorylated to form NADP + , which then receives hydrogen ions to form NADPH, a process that protects the reducing anabolic pathways from oxidative stress. Notably, NAD + is also a cofactor and substrate for hundreds of cellular enzymes and is one of the major contributors to maintaining cellular processes and ensuring cellular physiological functions [ 134 ]. In the early and middle stages of life, NAD + synthesis, metabolism, and consumption are in a balanced state. Specifically, NAD + is continuously utilised in cells by NAD + -consuming enzymes, including NAD + glycohydrolases, NADases (CD38, CD157, and Sarm1), and the protein deacetylase family of Sirtuins and poly ADP-ribose polymerases (PARPs), which participate in a variety of important cellular functions and generate the byproduct nicotinamide (NAM). To maintain intracellular NAD + levels, in the NAM recycling pathway, NAM is converted to NMN by nicotinamide phosphoribose transferase (NAMPT) and further converted to NAD + by nicotinamide mononucleotide adenosyltransferases NMNat1 (nucleus), NMNat2 (cytosolic face of the Golgi apparatus), and NMNat3 (mitochondria) [ 132 ]. In addition, NAD + can be synthesised from tryptophan via the kenuridine pathway and from vitamin precursors such as nicotinic acid via the Preiss-Handler pathway. Most tryptophan is metabolised to NAM in the liver and converted to NAD + via the NAM rescue pathway [ 135 ]. Thus, the NAM rescue pathway appears to be a major contributor to system-wide NAD + levels.

Under normal circumstances, NAD + is continuously decomposed, synthesised, and recycled to maintain the balance and stability of intracellular NAD + levels [ 136 ]. However, studies have found that the balance between NAD + catabolic and anabolic processes is altered during aging, with NAD + degradation rates exceeding the capacity for intracellular NAD + synthesis, or excess NAM being broken down by alternative intracellular metabolic pathways, effectively shifting it away from the NAM rescue pathway and further affecting NAD + levels [ 137 ]. Studies have demonstrated that when rodents or humans reach middle age, the level of NAD + in the body is reduced to half that at a young age, which severely impairs cellular energy metabolism and various biological pathways, accelerates the aging process, and increases the incidence of age-related diseases [ 138 ].

Relationship between NAD + loss and aging

NAD + levels are strongly associated with health and aging in both rodents and humans (Fig.  6 ). In 1937, scientists discovered that low levels of NAD + can lead to symptoms such as dermatitis, diarrhoea, and dementia. NAD + levels gradually decrease during aging, but the mechanism of this reduction is not fully understood. Recent studies have found that aging itself causes inflammation and oxidative stress, which affect the activity of NAMPT, the rate-limiting enzyme of NAD + synthesis, and further affect the activity of downstream NAD + -dependent enzymes (including Sirtuins, PARPs, CD38, and CD157) [ 135 ]. Notably, Sirtuins, PARPs, and CD38 are the main enzymes that consume NAD + , and their content and activity strongly affect intracellular NAD + level [ 139 ]. Sirtuins contain seven proteins (Sirtuin1–7) which are a class of NAD + -dependent deacetylases. They regulate the activity of various proteins and gene expression by consuming NAD + , and has been shown to be an important mechanism for regulating the life span [ 140 ]. PARPs activity is an important factor in intracellular NAD + catabolism. PARPs levels increase with age, possibly because DNA damage caused by aging requires PARPs enzymes to participate in repair; however, excessive activation of PARPs promotes the reduction of NAD + levels [ 141 ]. In addition, some studies have found that inflammation and SASP accumulation during aging promote the expression and activity of CD38 protein, leading to a partial reduction in NAD + levels and mitochondrial function through the regulation of SIRT3 [ 142 ].

NAD + metabolism and its relationship with aging. NAD + levels are maintained by three independent biosynthetic pathways. The kynurenine pathway uses the dietary amino acid tryptophan to produce NAD + . Tryptophan enters cells through the transporters SLC7A5 and SLC36A4. In the cell, tryptophan undergoes a series of reactions to form quinolinic acid, which is then converted by the quinolinic acid phosphoribosyl glycosyltransferase (QPRT) into nicotinamide mononucleotide (NAMN), where it converges with the Preiss-Handler pathway. In the Preiss-Handler pathway, niacin (NA) enters cells via SLC5A8 or SLC22A13 transporters, and is catalysed by the nicotinic acid phosphoribosyltransferase (NAPRT) to produce NAMN, which is then converted into NAD + by a series of reactions. The NAD + salvage pathway recycles the nicotinamide (NAM) generated as a by- product of the enzymatic activities of NAD + -consuming enzymes (sirtuins, poly (ADP- ribose) polymerases (PARPs) and the NAD + glycohydrolase and cyclic ADP- ribose synthases CD38, CD157 and Sarm1). Intracellular nicotinamide phosphoribotransferase (INAMPT) circulates NAM to nicotinamide mononucleotide (NMN), a portion of which enters the cell via SLC12A8 transporter and is then converted to NAD + by different NMNATs. Decreased levels of NAD + in cells during senescence give rise to a range of problems, including inflammageing, neurodegeneration, genomic instability (promoting senescence, apoptosis, and cancer), mitochondrial dysfunction, ROS accumulation, and loss of proteostasis

At present, many measures are used to inhibit NAD + consumption caused by aging or disease, including supplementation with various NAD + precursors such as nicotinamide mononucleotide (NMN) [ 143 , 144 , 145 ] and nicotinamide riboside (NR) [ 146 ], activation of NAMPT activity [ 147 ], and inhibition of CD38 activity [ 148 ]. Notably, Sirtuins, PARPs, and CD38 play active physiological roles in healthy cells. Thus, not every NAD + promotion strategy has a purely beneficial effect on the organism. Increasing NAD + levels by inhibiting PARPs activity reduces the ability of cells to repair DNA damage. Activation of Sirtuins enzyme expression objectively depletes NAD + , but also prolongs the lifespan of mice. In conclusion, to gain a more in-depth and comprehensive understanding of the effects of various NAD + promotion strategies, more clinical studies are needed to promote them for practical applications more safely, effectively, and scientifically.

Aging and disabled macro-autophagy

Autophagy is an indispensable part of cell metabolism that mediates the degradation and elimination of defective cellular components, including damaged nucleic acids, misfolded protein aggregates, abnormal lipids, and organelles, to promote homeostasis, differentiation, development, and survival through lysosomes [ 149 ]. Among the molecular phenotypic changes that occur during cellular aging, autophagy disorder has become an important physiological feature and has a causal relationship with aging-related diseases. Therefore, maintenance of an excellent autophagy process is essential for long-term health.

Cellular processes involved in autophagy

Autophagy is a highly conserved cell clearance pathway that targets macromolecules and organelles, and the integrity of its biological processes is related to the maintenance of cellular tissue homeostasis. Autophagy can be classified into three main types: macro-autophagy, micro-autophagy, and molecular chaperone-mediated autophagy. Specific target substances of autophagy can be divided into glycophagy and lipidophagy, mitochondrial autophagy, endoplasmic reticulum autophagy, nuclear autophagy, heterologous autophagy, and lysosomal autophagy [ 150 ]. These autophagy processes can be summarised as follows: expanded membrane structures (phagocytes) wrap some of the target material, such as defective organelles and misfolded protein aggregates, forming double-membrane sequestering vesicles (autophagosomes). Autophagosomes fuse with lysosomes and release their contents into the lysosomal lumen. The inner membrane of the autophagosome is degraded along with the encapsulated contents, and the resulting macromolecules are released into the cytoplasm for recycling via lysosomal membrane permeases [ 151 ]. Autophagy is a tightly regulated pathway that plays an important role in the regulation of basic metabolic functions, enabling cells to remove damaged or harmful components through catabolism and recycling and maintain the dynamic balance of nutrients and energy. Autophagy is also a major protective mechanism that allows cells to survive multiple stress conditions such as nutrient or growth factor deprivation, hypoxia, ROS, DNA damage, or intracellular pathogens [ 152 ]. In addition, autophagy is involved in many aging-related pathophysiological processes, such as tumours, metabolic and neurodegenerative diseases, and cardiovascular and pulmonary diseases [ 153 ].

Relationship between autophagy and aging

The stability or disturbance of autophagy has a causal relationship with health, aging and disease [ 154 ]. Increasing evidence indicates that intracellular lysosomal proteolytic function is impaired with aging in various model organisms, which impairs autophagic flux, exacerbates cell damage, and promotes the occurrence of aging-related diseases [ 155 ]. In both human clinical studies and rodent models, the expression of autophagy priming-related proteins ATG5-ATG12 and Becn1 decreased with increasing age, whereas the expression of mTOR increased [ 152 ]. The fusion rate of neuronal autophagosomes and lysosomes is decreased in aged mice, and neuronal autophagy is reduced, which further leads to the appearance of misfolded, mislocalized, and aggregated proteins in the nervous system and increases the probability of neurodegeneration [ 156 , 157 ]. These findings suggest a causal relationship between impaired autophagy and aging [ 158 , 159 ]. This conclusion was confirmed in animal models by manipulating key genes regulating autophagy. Thus, an increase in autophagy caused by heredity, gene mutation, or pharmacological intervention can prolong the life of animals. Studies in C. elegans have found that daf-2 inactivation mutations are dependent on autophagy genes, such as bec-1 , lgg-1 , atg-7 , and atg-12 , and that this mutation significantly extends the lifespan of C. elegans [ 160 ]. Enhanced autophagy in aging mice can also activate mitochondrial SIRT3, inhibit oxidative stress and maintain immune memory [ 161 ]. Accordingly, researchers have found more damaged autophagy sites in aging model animals, which manifest as reduced autophagosomes and impaired lysosome fusion or degradation ability, accompanied by the accumulation of abnormal organelles or biological macromolecules in the cell, leading to cell dysfunction and even death. This eventually increases the incidence of age-related diseases, such as neurodegenerative, heart, and metabolic diseases [ 162 ]. Although the important role of autophagy in inhibiting aging and prolonging lifespan has been widely confirmed, excessive upregulation of autophagy under certain physiological conditions may also cause cell metabolic disorders. For example, the overexpression of Rubicon, a negative regulator of autophagy in aged mice, disrupts adipose metabolism in tissue cells, and the lack of serum/glucocorticoid-regulated kinase-1 (sgk-1) leads to increased mitochondrial permeability and enhanced autophagy, which further leads to reduced environmental adaptability in C. elegans and mice [ 163 , 164 ].

In conclusion, aging is accompanied by a decline in autophagy. Enhancing the autophagic ability in aging model animals is essential for maintaining homeostasis of cell metabolic function, prolonging life span, and improving pathological aging and diseases. Concurrently, autophagy is also one of the important regulators in the aging process. Enhancing or restoring autophagy function to a certain extent is beneficial to the health and longevity of various animal models, whereas dysregulation of autophagy in any direction, whether too low or too high, leads to cell defects and a decline in body function.

Aging and stem cell exhaustion

Physiologically, the decline in stem cell regenerative ability is closely related to the degree of senescence, which is manifested in the accumulation of global harmful cell metabolites caused by aging, and also impairs the regenerative ability of stem cells. Conversely, stem cell decline is an important cellular driver of a variety of tissue senescence-related pathophysiologies.

Main causes of stem cell exhaustion

Stem cells are progenitor cells with the potential for self-replication and multidirectional differentiation. Through self-renewal and differentiation, they can produce mature effector cells, replenish and repair damaged organs, and maintain the health and vitality of the human body; thus, they promote a steady state of continuous organisation throughout the life course [ 165 ]. Numerous studies have demonstrated that stem cells play an irreplaceable role in different stages of life. During the growth and development stages, stem cells continue to differentiate into a variety of new cells for growth and development [ 166 ]. During adulthood, stem cells replace senescent or damaged cells to maintain normal physiological metabolism of the organism [ 167 ]. Notably, throughout the life cycle, stem cells can recognise the signals released by aging damaged cells in the body, localise to the place that needs repair and regeneration, and differentiate into cells at that location to achieve an overall improvement of body functions. However, during the aging process, the proportion of stem cells to total cells gradually decreases. The proportion of mesenchymal stem cell cells in the bone marrow during aging is reportedly 200 times lower than that at birth [ 168 ]. Numerous studies have shown that during aging, a series of changes occurs in the tissues and cells of an organism, including increased DNA damage, replication stress, loss of polarity, mitochondrial dysfunction, altered autophagy, and epigenetic disorders, all of which contribute to stem cell aging and exhaustion [ 20 , 21 , 169 , 170 ]. In addition, the stem cell microenvironment (also called “Niche”) plays a crucial role in maintaining and regulating stem cell function and tissue homeostasis [ 171 ]. During aging, stem cells also accumulate in large quantities as the niche changes, and functional differences between “young and old” stem cells can be more dependent on mechanical differences in the stem cell niche, rather than cell-autonomous age-related changes [ 172 ]. This is also risk for stem-cell injection treatments, as the niche itself may need rejuvenation prior to fresh stem cells.

Although stem cells are not affected by replicative senescence, they are still susceptible to damage and accumulate in large quantities during the aging process. Based on the importance of stem cells on the basis of cell lineages, their dysfunction may have a greater impact than that of other cell types [ 173 ]. As aging progresses, stem cells tend to accumulate DNA damage, which reduces their ability to regenerate cell lineages, exhibiting age-related loss of organ function and homeostasis, and increasing the incidence of age-related diseases [ 174 ]. However, little is known about the cause of this damage or the mechanism by which it leads to a decline in senescent stem cell function. In some cases, DNA damage can lead to stem cell apoptosis, aging, and differentiation, thereby reducing stem cell numbers. Studies have also shown that increased ROS levels in aging mesenchymal stem cells and increased ROS expression in haematopoietic and neural stem cells in mice lead to abnormal cell proliferation, tumour-like changes, and decreased self-replication of stem cells [ 175 ]. Similarly, dysregulation of autophagy during aging leads to defects in protein homeostasis, impaired protein folding, and the accumulation of toxic proteins, resulting in cell damage and tissue dysfunction, and stem cells can also be damaged or depleted [ 176 , 177 ]. Increased mitochondrial DNA point mutations and deletions, along with a shortened lifespan and premature aging, result in decreased nutrient uptake by stem cells. Conversely, enhanced mitochondrial function is accompanied by enhanced stem cell function and tissue regeneration [ 178 ]. Epigenetic regulation also plays an important role in the regulation of stem cell function and changes in the epigenome during aging affect the aging process of stem cells [ 179 , 180 ]. DNA methyltransferases balance self-renewal and differentiation in multiple adult stem cell compartments [ 181 ]. Conditional knockout of DNA methyltransferases results in reduced proliferation, abnormal differentiation, and impaired self-renewal of stem cells [ 182 ]. In addition, proper histone modification is necessary for stem cell self-renewal, and the activity of histone acetyltransferases is important for maintaining the homeostasis and function of neural stem and progenitor cells [ 182 , 183 ]. In summary, stem cells are the source cells of organism renewal, and their function is affected by many microenvironmental factors in senescent cells; therefore, stem cell senescence is closely related to the drivers of aging, health, and longevity.

Relationship between stem cell exhaustion and aging

During the aging process, a decrease in stem cell number and function is closely related to a decline in tissue function and repair ability. In recent years, with the development of new molecular techniques, such as single-cell transcriptomics, lineage tracing, and clonal analysis, scientists have discovered the commonality and heterogeneity of stem cell senescence across tissues. Particularly, the ability of stem cells to produce offspring is impaired during aging. The number of activated neural stem cells and mature nerve cells (offspring) decreases with age, with older haematopoietic stem cells producing fewer lymphoid cells that are dynamically activated and differentiate more slowly [ 184 ]. The fate and behaviour of stem cells in senescent tissues are abnormal, and they may be in a senescent, over-activated, or abnormal differentiation state [ 185 ]. In addition, somatic cells in the stem cell pool are more susceptible to mutations and clonal competition during aging, while the heterogeneity of the resting stem cell pool increases, and the ability to produce established offspring decreases [ 186 ]. Specific age-related transcriptomic and proteomic markers accumulate in senescent stem cells and induce the infiltration of different types of immune cells into the stem cell microenvironment. Researchers have also found that clonal expansion of T-and B-cell infiltrates occurs in the aging brain tissue of mice and humans, and this infiltrate is more pronounced in age-related diseases [ 187 , 188 ].

In summary, stem cells, as primitive and undifferentiated cells, possess a strong regenerative capacity and are essential for environmental homeostasis and organ regeneration in mammalian tissues. However, the number of stem cells continues to decrease with age, and their ability to self-renew and differentiate decreases, leading to impaired tissue or organ regeneration. Therefore, stem cell senescence is closely associated with aging. Because of the important role of stem cells in maintaining functional homeostasis during aging, they have attracted considerable attention in the fields of disease therapy, regenerative medicine, and new drug development.

Aging and cellular senescence

Senescent cells in tissues and organs are thought to be essential not only for the aging process but also for the onset of chronic diseases [ 189 ]. During aging, cells exposed to metabolic, genotoxic, or oncogene-induced stress undergo a basically irreversible cell cycle arrest called cellular senescence [ 190 ]. A major phenotype of senescent cells and how they are thought to promote disease is an increase in inflammatory mediators, mainly cytokines and chemokines, known as the SASP, it causes dynamic equilibrium damage by interfering with stem cell regeneration, tissue and wound repair, and inflammation [ 191 ]. As the number of senescent cells increases with age, cell senescence has been associated with several age-related diseases, the elimination of senescent cells with drugs for aging may be an effective treatment for several previously untreatable diseases [ 192 ].

Main causes of cellular senescence

Cell senescence is a kind of cell state caused by stress injury and some physiological processes, which is characterized by irreversible cell cycle arrest, accompanied by secretory features, macromolecular damage and metabolic changes, these functions can depend on each other to jointly drive the aging process [ 193 ]. Cell senescence may be an alarmist response to deleterious stimuli or aberrant proliferation, including cell cycle exit quiescence and terminal differentiation. Quiescence is a state of temporary arrest in which proliferation can be restored with appropriate stimulation; terminal differentiation is the acquisition of specific cellular functions, accompanied by persistent cell cycle arrest mediated by pathways distinct from cellular senescence [ 194 ]. In senescent cells, the cyclin-dependent kinase (CDK2) inhibitor P21 WAF1/CIP1 (CDKN1A) and the CDK4/6 inhibitor p16 INK4A (CDKN2A) accumulate, this accumulation leads to sustained activation of retinoblastoma (RB) family proteins, inhibition of E2F transactivation, and subsequent cell cycle arrest [ 195 ]. ARF (an alternative reading frame protein at the P16 INK4A gene locus that activates p53) has an important role in regulating cell cycle arrest [ 196 ]. In addition, cell cycle arrest is also characterized by defects in ribosome biogenesis and retrotransposon [ 197 ]. Senescent cells secrete a number of cytokines, including proinflammatory cytokine and chemokines, growth regulators, angiogenic factors and matrix metalloproteinase, collectively known as the SASP or the senescence information secretory group. SASP has been recognized as a marker of senescent cells and mediates many pathophysiological effects [ 198 ]. In addition, DNA damage, telomere depletion, epigenetic changes, protein damage, lipid damage, dysfunction of mitochondria and lysosomes, ROS and inflammation are all important inducers of cell senescence [ 199 ].

Relationship between cellular senescence and aging

Aging is a complex biological process, which is closely related to cell function [ 200 ]. Over the past few decades, a growing body of research has found that cellular senescence is a key driver of many age-related diseases. Excessive accumulation of senescent cells may lead to many chronic diseases and accelerate organ aging. This process not only affects health, but also promotes mutual cellular and physical aging.

Senescent cells exhibit abnormally high levels of damage accumulation, including DNA damage, telomere dysfunction, mitochondrial dysfunction and ROS accumulation [ 201 ]. Elimination of senescent cells reduces the number of cells with the highest degree of damage [ 191 , 198 ]. Therefore, treatment methods that improve or delay cellular senescence characteristics are important strategies for delaying aging. Studies found that after clearance of senescent cells, lower levels of telomere-associated foci were detected in aortic epithelial and hepatocytes of aging mice [ 202 , 203 ]. Similarly, after genetic or pharmacological clearance of senescent cells in mice with chemotherapy-or whole-body irradiation-induced senescence, a reduction in cells bearing persistent DNA damage was observed [ 204 ]. Notably, after clearance of senescent cells, the above evidence relates only to DNA damage, and no data currently provide insights into protein or lipid damage or other senescence-associated phenotypes after clearance of senescent cells.

A new class of drugs, “Senolytics,” eliminate senescent cells by inhibiting a targeted pathway that ultimately damages cell apoptosis [ 205 ]. The senolytic approach aims to selectively eliminate senescent cells, with a pioneering study showing that about 30% of senescent cells are cleared and that heart, kidney and adipose tissue function is improved [ 206 , 207 ]. Subsequent research has focused on specific age-related diseases such as frailty, Idiopathic pulmonary fibrosis, arteriosclerosis, osteoporosis, liver steatosis, and osteoarthritis, in these cases senescent cell clearance has been shown to be beneficial [ 208 ]. Notably, elimination of senescent cells has been shown to alleviate age-related diseases, but not necessarily successfully delay aging. Aging is generally considered a negative phenomenon, but in the context of disease-free aging, senescent cells can retain at least part of their pre-senescent phenotype and function [ 209 ]. The elimination of senescent cells will also result in the filling of the empty space by new cells, which requires the proliferation of stem cells or other resident cells, which may lead to the depletion of their regenerative potential and replication of senescence [ 198 ]. Cellular senescence appears to be a trade-off between tissue function and the risk associated with injury accumulation. Therefore, the therapeutic strategy of intermittent short-term clearance of senescent cells provides a new perspective to solve this problem. In summary, the rate at which cellular damage accumulates determines the rate at which cells senescence, and implies the rate at which the aging process occurs in a disease/healthy state. It is reasonable to understand that proper elimination of senescent cells may be an effective strategy to control aging-related diseases and delay aging.

Other possible causes of aging

Aging is a complex biological process characterised by age-related adaptive decline and a decline in organic physiological systems and metabolic pathways. In addition to the major contributors to aging that we have reviewed, factors such as inflammation, loss of epigenetic information, resurrection of endogenous retroviruses, loss of protein balance, deregulated nutrient sensing, altered intercellular communication, and tissue dysbiosis are also important drivers of accelerated aging.

Many studies have demonstrated that chronic inflammatory response activates the nuclear factor (NF)-κB signalling pathway, a key intracellular signalling pathway for inflammation, which then influences the fate of tissue cells towards senescence by regulating the downstream mechanistic target of rapamycin (mTOR) pathway, insulin and insulin-like growth factor pathways, the AMPK, Sirtuin, forkhead box O families, and p53-related pathways [ 12 , 210 , 211 , 212 , 213 ]. The loss of epigenetic information is also an important cause of aging in mammals. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging [ 214 ]. It was reported the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation [ 215 ]. The latest research has found that the resurrection of endogenous retroviruses is a hallmark and driving force of cellular senescence and tissue aging. Activation of endogenous retroviruses has been observed in organs of elderly primates and mice, as well as in human tissues and serum of elderly individuals. Their inhibition alleviates cellular aging and tissue degradation, and to some extent, alleviates the aging of the body [ 216 ].

In addition, a decline in the activity of the protein-folding chaperone network and loss of intracellular protein balance are important markers of aging [ 217 ]. Decreased Sirtuin1 and HSP70 levels in senescent cells impair protein homeostasis and heat shock response [ 22 , 218 ]. A growing body of literature has also shown that the ubiquitin-proteasome system is the main non-lysosomal pathway by which cells control protein degradation, either by promoting central lifespan regulators or by aberrant folding and degradation of damaged proteins, and it plays a potential role in regulating the aging process [ 219 , 220 ]. Aging-induced telomere shortening, mitochondrial dysfunction, and DNA damage affect cellular nutrient perception [ 221 , 222 , 223 ]. Aging is also associated with progressive changes in cell-to-cell communication, including factors in the blood-borne system that promote aging or prolong life, interaction of different communication systems between cells, and interference of two-way extracellular matrix communication during aging [ 223 ]. Among the blood-derived factors with pro-aging effects, chemokines CCL11, eosinophil granulocyte, and inflammation-related protein b2-microglobulin can reduce neurogenesis, IL-6 and transforming growth factor β can inhibit the haematopoietic system, and complement factor C1q can affect muscle repair [ 25 , 26 ]. Notably, these blood-derived factors are secreted in the context of SASP, and may contribute to the “infectious” aging phenomenon. Moreover, it has been demonstrated that the anti-aging blood transmissible factors in the blood of young mice can effectively restore the renewal and repair ability of old mice, and reduce the expression of age-related genes [ 224 ]. Cell-to-cell communication also involves short-term从 n extracellular molecules, including ROS, nitric oxide, nucleic acids, prostaglandins, and other lipophilic molecules [ 25 ]. The interactions between soluble factors released from different tissues may also play a role in pro- or anti-aging effects during the aging process. In essence, all of the above-mentioned causes of senescence can lead to dynamic equilibrium disorder within the cell, thus providing a stable proliferative arrest response to various stressors -- cellular senescence. Although senescence promotes programming during development and wound healing, it also limits tumor initiation. However, dynamic equilibrium disorders within senescent cells and their production of large amounts of SASP will induce an inflammatory state that triggers local and systemic inflammation and tissue damage. The pathological accumulation of senescent cells is also associated with a range of diseases and age-related diseases across the organ system. In preclinical and clinical models of aging and chronic diseases, therapeutic approaches that induce apoptosis of senescent cells or inhibit the senescence-associated secretory phenotype have been shown to be pharmacological targets for delaying systemic aging in the body.

Changes in the gut microflora during aging have also attracted great interest from scientists. The gut microbiota is involved in many physiological processes, such as digestion and absorption of nutrients, protection from pathogens, and production of essential metabolites, including vitamins, amino acid derivatives, secondary bile acids, and short-chain fatty acids [ 225 ]. The gut microbiota also signals to peripheral and central nervous system organs and other distant organs and has a strong impact on the overall maintenance of host health [ 226 ]. The interruption of the two-way communication between bacteria and the host leads to biological disorders and causes various pathological conditions, such as obesity, type 2 diabetes, ulcerative colitis, neurological disease, cardiovascular disease, and cancer [ 28 , 227 ]. In addition, vitamin D and magnesium deficiency is associated with aging-related diseases [ 228 ]. Vitamin D aids in magnesium absorption, and magnesium helps in the synthesis and activation of vitamin D in the body [ 229 ]. This interaction reduces the formation of age-related insoluble proteins, and a deficiency of either vitamin D or magnesium can affect muscle, bone, nerve and heart health.

In summary, many studies have linked biological processes such as telomere dynamics, DNA damage response, mitochondrial dysfunction, NAD + loss, autophagy dysregulation, stem cell exhaustion, inflammation, loss of protein balance, dysregulation of nutrient sensing, changes in cell-to-cell communication, and dysbiosis to the triggers behind the characteristics of aging. Aberrant perturbations in these biological processes create feedback loops that amplify the aging phenotype, accelerate the aging process, and increase the incidence of age-related diseases. However, in the early stage of development, the above-mentioned physiological characteristics of aging to a certain extent also promote the healthy development of juvenile factors. For example, the activation of nutrient-sensing signals during early development contributes to organ development in adolescents; and the activation of nutrient-sensing signals after aging has a largely pro-aging effect [ 230 , 231 ]; Low-dose mitochondrial dysfunction can stimulate cells to engage in a beneficial antagonistic response through mitosis [ 232 ]; appropriate levels of cellular senescence contribute to inhibition of tumour generation and promote wound healing [ 233 ]. Therefore, it is an important prerequisite to understand the physiological characteristics of aging and its molecular mechanism in specific life stages and physiological states.

• Anti-aging strategies

The interaction and mutual promotion between aging triggers, aging phenotypic characteristics, and inherited or acquired age-related diseases have become key hotspots of interest for scientists to explore anti-aging strategies. To date, there have been a wide range of interventions aimed at mitigating aging or aging-related morbidity, including dietary regulation and caloric restriction, improved sleep quality, enhanced physical activity, altered microbiota, and exogenous active molecular interventions targeting specific senescence-promoting molecular targets.

The role of diet and calorie-restriction in delaying aging

Diet is strongly correlated with longevity and disease development. In rodents and humans, the excessive intake of high-calorie foods increases body fat production, fat storage, and obesity. People with obesity are more likely to develop symptoms such as elevated insulin, blood sugar, cholesterol, and triglyceride levels during aging, and a combination of these factors can activate the aging pathway and accelerate the aging process, leading to disease and death [ 234 ]. Studies based on diet regulation and calorie restriction have shown that moderate calorie restriction in mice modestly extends the lifespan and improves metabolic, cerebrovascular, and cognitive function indices [ 235 , 236 , 237 ]. Dietary restriction improves cognition and reduces plaque burden in mice with Alzheimer’s disease through a mechanism related to mitochondrial function in hippocampal neurons [ 238 ]. Researchers recently found that controlling the intake of nutrients, including total calories and macronutrient balance, had a greater effect on aging and metabolic health than the three commonly used life-extension drugs (metformin, sirolimus, and resveratrol) [ 239 , 240 ]. A recent study also showed that moderate caloric restriction can reduce the production of acidic and cysteine-rich (SPARC) proteins, which are associated with aging, and extend the healthy lifespan of older people, further confirming that improving the diet is an important way to live a long and healthy life [ 241 ].

The role of sleep quality in delaying aging

Sleep is an important factor in the recovery and improvement of physiological systems, including metabolism, endocrine function, immune response, and brain metabolism. Poor sleep accelerates aging and increases the incidence of age-related diseases, such as cognitive decline, Alzheimer’s disease, haematopoietic stem cell dysfunction, and coronary heart disease. Numerous studies have reported that improving the quantity and quality of sleep can be considered as an anti-aging treatment that can prevent, slow, or even reverse the physical decline and degeneration associated with the aging process [ 242 ]. One clinical trial reported a significant association between better quality and quantity of sleep and increased plasma levels of S-Klotho, a gene family known as a senescence suppressor that is overexpressed to prolong life [ 243 ]. In addition, researchers followed 411 volunteers for eight years and found that poor sleep quality may be a new modifiable risk factor for coronary heart disease in older adults, independent of traditional cardiovascular risk factors [ 244 ]. In human trials, adequate sleep has been found to regulate the epigenome of haematopoietic stem and progenitor cells, inhibit inflammatory output, and maintain clonal diversity to slow the decline in the hematopoietic system [ 245 ]. Sleep and health undergo age-related changes throughout life, and the impact of sleep deprivation in older people is particularly important [ 246 , 247 , 248 ]. However, to improve sleep quality in the older people and reduce age-related sleep issues, further research is needed to investigate the relevant mechanisms.

The role of exercise in delaying aging

Physical activity causes a series of integrated physiological responses in many tissues in the entire animal kingdom and has been widely accepted to improve the health of physiological tissues. Exercise is strongly associated with changes in plasma microsecretory factors, such as immunomodulatory cytokines, regulatory T cells in lymphoid organs, and inflammatory monocytes. Increased physical activity in aged mice significantly slowed cognitive aging and neurodegeneration, and these improvements were associated with the reduced expression of neuroinflammatory genes in the hippocampus. Proteomics of plasma from exercising mice has revealed a significant increase in the complement cascade inhibitor rapamycin, which binds to brain endothelial cells and reduces the expression of neuroinflammatory genes in mice with acute encephalitis and Alzheimer’s disease [ 249 ]. In addition, exercise can induce hippocampal precursor cell proliferation in aged mice by activating platelets, that is, increasing the systemic levels of the platelet-derived exerkine CXCL4/platelet factor 4 (PF4) ameliorates age-related regenerative and cognitive impairments in a hippocampal neurogenesis-dependent manner [ 250 ]. Although the benefits of exercise have been demonstrated in several studies, older people are less likely to exercise if they are physically weak or have poor health status. Based on this, researchers were able to ameliorate age-related neurogenesis and cognitive impairment in the hippocampus in the older people by systematically administering plasma from exercising mice, and the key molecular target of this regulation was identified; exercise increased the concentration of liver-derived glycosylphosphatidylinositol-specific phospholipase D1 in the plasma of aged mice [ 251 ]. Similarly, many studies have found that exercise can enhance myocardial contractility [ 252 ], improve heart pumping function and heart ejection fraction [ 253 ], improve blood supply [ 254 ] and oxygen supply [ 255 ] in all organ systems of the body, and speed up metabolism [ 256 ], prolonging the lifespan of cells, thereby slowing the aging process of organs and skin.

The role of exogenous active molecules in delaying aging

Lifestyle changes, including calorie restriction, sleep regulation, and exercise, are insufficient to extend the healthy lifespan of older people or prevent age-related diseases. Therefore, many studies have focused on the mechanisms underlying the aging process and explored ways to target the hallmark features of aging. Currently, the most promising mechanisms for preventing senescence include inhibition of the mTORC1 signalling pathway [ 257 ], clearance of senescent cells [ 258 ], and the use of natural metabolites to rejuvenate stem cells [ 221 ]. Therefore, the development of synthetic or natural small-molecule compounds that inhibit these signature features is a promising anti-aging strategy.

To date, many synthetic or natural small-molecule compounds have been reported to have the potential to genetically protect or regulate senescence in one or more model species (Table  1 ). Among them, studies on synthetic compounds including metformin, klotho, PF4, hyaluronan acid, taurine, acarbose, rapamycin, spermidine, NAD + enhancers, nonsteroidal anti-inflammatory drugs, lithium, reverse transcriptase inhibitors, systemic circulating factors, glucosamine, glycine, and 17α-oestradiol, focused on telomere attrition, DNA damage, mitochondrial dysfunction, NAD + loss, disabled macro-autophagy, stem cell exhaustion, regulation of tissue cell perception of nutrients, cell-to-cell communication, and improved stem cell function. By improving the interactions among the hallmarks of aging, these synthetic compounds can eventually alleviate or even reverse the decline in age-related bodily functions.

Senescent cells cannot continue to divide or die in tissues, and secrete a range of pro-inflammatory factors that may recruit inflammatory cells to reshape the extracellular environment, induce abnormal cell death and fibrosis, and inhibit stem cell function [ 258 ]. Senescent cells are also closely related to the pathogenesis of osteoporosis, atherosclerosis, hepatic steatosis, pulmonary fibrosis, and osteoarthritis. Therefore, anti-aging methods targeting senescent cells are also very important anti-aging strategies and can be divided into two categories: senescent cell lytic agents (senolytics), whose role is to clear senescent cells, and compounds that combat the effects of various cytokines secreted by senescent cells. Currently, senolytics class targeting senescent cells and SASP include natural polyphenol extract, kinase inhibitors, BCL-2 family inhibitors, heat shock protein inhibitors, BET family protein degraders, P53 stabilizers, cardiac steroids, PPRα agonists and antibiotics.

Other anti-aging strategies

Heterochronic blood exchange models have shown that the blood of aging mice accelerates the aging of tissues and cells in young mice [ 304 , 305 ]. Based on this, the researchers showed that systemic exposure of aged male mice to a fraction of blood plasma from young mice containing platelets decreased neuroinflammation in the hippocampus at the transcriptional and cellular level and ameliorated hippocampal-dependent cognitive impairments [ 263 ]. Despite the growing number of reports about heterochronic blood exchange research, it still faces great challenges and risks in the field of anti-aging, both at the technical level and in the application of safety. In addition, mesenchymal stem cell therapy has been shown to improve frailty and facial skin aging [ 306 ]. Intravenous mesenchymal stem cell may be an effective treatment for frailty in the elderly. However, the safety and efficacy of stem cell therapy remain controversial, and more studies are needed to verify it. Studies have found that deliberate cold exposure can enhance the nervous system, as well as injury and speed recovery [ 307 ]. Saunas are effective at activating cell longevity and anti-cancer factors through heat, this may be a useful tool for people who are too old to engage in physical activity [ 308 ], but its physiological metabolic mechanisms and safety thresholds remain to be further confirmed.

Conclusions and perspectives

The physiological characteristics of aging summarised in this article gradually accumulate over time and contribute to the aging process. Notably, antagonism of an organism’s response to the characteristics of aging also plays a subtle role in the aging process. When the cumulative damage caused by primary and antagonistic markers is no longer compensated for by the complex markers of aging, it means that the rate of aging is accelerated. Furthermore, senescence also relies on the integration of cell-autonomous and non-cell-autonomous mechanisms, and mechanisms that promote senescence can be transmitted between different types of organs and cells. In a metachronous experiment linking the vasculature of young and old mice, extensively characterised by single-cell transcription levels, a spatiotemporal map of the ability of the young system to rejuvenate the senescent system was confirmed, and vice versa; the factors that promote aging have the ability to accelerate the aging of young cells. This may explain why programmed aging usually affects multiple organs in a nearly synchronous manner, causes a comprehensive systemic decline in physiological function, and is an important factor that induces pathological aging.

In conclusion, aging is a gradual and complex process of decline in physiological function, and experiments in animal models have shown that certain interventions may not only extend lifespan, but also increase healthy longevity. However, in vitro models, tissue culture studies, and in vivo animal models, which are ultimately translated into human studies, are complex and diverse, and only a few models can be used to investigate these differences. There are also significant differences between physiological and pathological aging, and the scientific problem of slowing down aging and extending the healthy lifespan of humans involves a number of challenges, including inadequate regulation, barriers to clinical validation, failure to identify more biomarkers of human aging, and the unknown challenges of introducing new interventions to the market. It is gratifying that years of basic research in the anti-aging field have laid the foundation for explosive biotechnology and industrial applications. In a recent report, researchers used a “visual genetic circuit” to control two pathways of yeast aging, alternative approaches including the lysine deacetylase Sir2-related pathway and the haeme-activating protein (HAP)-related pathway, successfully extending yeast lifespan by 82% [ 309 ]. Hence, using modern biological techniques, including genetic manipulation or cell-based therapies with broad implementation prospects, to focus on the discovery of physiological mechanisms and interventions underlying the aging process will greatly advance anti-aging research, delay human aging to the maximum extent, maintain human physiological functions in later years, and mitigate the surge in age-related chronic diseases.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

Nicotinamide adenine dinucleotide

DNA damage response

Telomerase reverse transcriptase

Telomerase RNA component

Reactive oxygen species

Senescence-associated secretory phenotype

Adenosine triphosphate

Adenosine 5’-monophosphate

Tricarboxylic acid

Nicotinamide

Nicotinamide phosphoribose transferase

Poly ADP-ribose polymerases

5'-AMP-activated protein kinase

Platelet factor 4

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This work was supported by Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-CXRC-031).

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Yumeng Li, Xutong Tian, Juyue Luo, Tongtong Bao & Xin Wu

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Yumeng Li wrote the manuscript. Xutong Tian, Juyue Luo and Tongtong Bao prepared the figures. Shujin Wang reviewed the manuscript. Xin Wu offered the idea of the manuscript. All authors reviewed the manuscript.

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Li, Y., Tian, X., Luo, J. et al. Molecular mechanisms of aging and anti-aging strategies. Cell Commun Signal 22 , 285 (2024). https://doi.org/10.1186/s12964-024-01663-1

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