2.4.1 DNA editing

CRISPR–Cas systems target particular DNA sequences with the assistance of guide RNA, which recognizes the protospacer adjacent motif (PAM).²⁸ Of the two naturally existing CRISPR–Cas immune systems, the class 1 (types I, III, and IV) system applies multiprotein assemblies to split nucleic acids, whereas the class 2 systems (types II, V, and VI) deploys a single protein effector domain for cleavage.²⁹ Notably, Cas9, Cas12, and Cas13 in the class 2 system are currently the subjects of investigation.³⁰ For gene targeting and genome editing applications, class 2 CRISPR-associated nucleases serve as adaptable tools for nucleic acid detection and handling.³¹

2.4.2 Base editing

2.4.3 Prime editing

Prime editing (PE) represents an adaptable and accurate genome modification method, enabling the direct incorporation of new genetic material at a targeted DNA locus. This technique employs a catalytically hindered Cas9 endonuclease combined with a custom-designed reverse transcriptase guided by a PE guide RNA (pegRNA) to achieve its editing objectives.⁴⁸ PE works in various cells, organoids, and mice embryos with varying degrees of success.^48-50 PE2 rescued the expression of an mRNA/long noncoding RNA gene pair by precisely editing a single base at a transcription factor binding site, thus broadening the genome editing toolkit.⁵¹ Chen et al.⁵² developed PE4 (PE2 plus a dominant negative mismatch repair protein), PE5 (PE3 plus a dominant negative mismatch repair protein), and PEmas (synergy with PE4, PE5, and engineered pegRNAs) systems, greatly enhancing PE efficiency and outcome purity at numerous inherent genomic locations in mammalian cells. Recently, split PE has become increasingly popular. Zheng et al.⁵³ successfully delivered a dual-AAV8 split prime editor into mice. Liu et al.⁵⁴ improved the method by splitting pegRNAs into a single guide RNA (sgRNA) and a circular RNA RT template, thereby boosting its adaptability and stability.

PE has manifested superior or equivalent efficiency to HDR while producing fewer byproducts. It presents a distinct array of advantages and drawbacks compared with base editing. Moreover, it incurs substantially fewer off-target modifications than Cas9 at identified Cas9 off-target positions⁴⁸ and provides more targeting versatility.^{30, 55} PE also enables the effective generation of tiny deletions and insertions, expanding its applicability to almost 90% of disease-associated mutations.⁵⁶

Gene editing techniques provide accurate tools for gene insertion, deletion, and rectification.⁵⁷ CRISPR–Cas genome editing systems have revolutionized life science research and driven medicine translation.⁵⁸ The advancement of gene therapy has relied on multiple pivotal factors, such as the refinement of gene delivery methods, comprehension of the genetic origins of diseases, creation of novel nucleic acid-based therapies, and innovation in genome engineering technologies.⁵⁹ Improvements in the stability, safety, and pharmacokinetic profiles of gene delivery vectors are instrumental in evoluting gene therapy.⁵⁹

3.1.1 Retroviruses and lentiviruses

Retroviruses and lentiviruses, both enveloped single-stranded RNA viruses,⁶² share a similar genomic structure. Their genomes contain the env, gag, and pol genes flanked by long terminal repeats, which harbor enhancer/promoter elements necessary for integrating the host cells’ genome via double-stranded DNA (dsDNA) provirus formation.⁶³ Retroviruses can package approximately 8 kb of exogenous sequences.⁶³ They execute reverse transcription, converting their RNA genome into dsDNA replicas. These DNA copies can subsequently be integrated into the host genome.⁶⁴ However, retrovirus vectors can only transduce dividing cells, posing a significant hurdle for gene therapy applications.⁶⁵ In contrast, lentivirus vectors are capable to transduce dividing and nondividing mammalian cells,⁶⁶ making them an attractive tool for permanent genome modification.⁶⁷ Clinical trials have underscored the therapeutic effectiveness of lentiviral vectors in various diseases, including ocular disorders. Lentivirus vectors have shown their capacity to transduce various ocular tissues following intravitreal injection, including retinal pigment epithelium (RPE), trabecular meshwork, corneal endothelium, ciliary body, anterior chamber, iris pigmented epithelium, inner nuclear layer, and ocular muscles,⁶⁸ with subretinal injection being safe, tolerable, with no dose-constraining toxicities, mild ocular inflammation, and reproducible, prolonged transgene expression.⁶⁹ In ocular gene therapy, both retroviruses and lentiviruses have been utilized in the development of RetinoStat for treating neovascular AMD (ClinicalTrials.gov Identifier: NCT01301443 and NCT01678872). Continuous monitoring of individuals undergoing lentiviral vector-mediated gene therapies remains essential to assess long-term safety and efficacy. Despite progress, further basic and clinical research is still required to enhance production and transduction efficiency.

3.1.2 Adenoviruses

Adenoviruses are nonenveloped, linear, dsDNA viruses with genomic sizes typically around 40 kb.⁷⁰ Their wide range of host cell tropisms enables them to infect host cells regardless of their cell division status.⁷¹ Unlike retroviruses, adenovirus infection is independent of the cell-cycle phase and does not involve integrating viral genes into the host genome.⁷² Human adenoviruses encompass over 100 subtypes (serotypes 1−52 and genotypes 53−103).⁷² Engineered adenovirus vectors have found applications in gene therapy, cancer therapy, and vaccine development.^73-75 Clinical trials of adenoviral gene therapy have revealed mild inflammatory response, the capability to infect different cells, and a low risk of chromosome mutagenesis, offering considerable safety and effectiveness in in vivo gene therapy.^{76, 77} Adenoviral vectors are extensively utilized in gene therapy research, with notable progress observed in in vivo and ex vivo genome editing, independently or in combination with other vectors, despite facing certain challenges.^{78, 79}

3.1.3 Adeno-associated viruses

AAV is a small nonenveloped, single-stranded DNA (ssDNA) genome encapsulated within an icosahedral capsid, about 22−26 nm in diameter.⁸⁰ It comprises a 4.7 kb ssDNA genome flanked by inverted terminal repeats (ITRs). These ITRs, spanning 145 nucleotides, can self-anneal into hairpin forms and are essential for genome encapsulation following replication and function as packing signals.⁸¹ AAV1–AAV13 represent 13 distinct serotypes found in human and nonhuman primates.⁸² AAV genome is characterized by its highly condensed structure, incorporating coinciding coding regions, alternative splicing patterns, and multiple translation initiation sites derived from canonical and noncanonical start codons.⁸⁰ rAAVs are the most prevailing gene delivery vectors,⁸³ produced by replacing viral sequence with a transgenic cassette.⁸⁴ The capsid, genome, and transgene product are AAV vectors’ primary immunogenic components,⁸⁵ with intravitreal administration inducing dose-dependent inflammatory responses.⁸⁵ Despite this, AAVs exhibit lower immunogenicity than other viral vectors.⁸⁶ AAV-based gene therapy has exhibited safety and sustained effectiveness in several preclinical trials.^{83, 85} Figure 3 illustrates AAV vectors’ transduction mechanism.

4.1.1 Retinitis pigmentosa

RP, or rod-cone dystrophy, represents a heterogeneous group of hereditary diseases featured by the initial loss of rod photoreceptor cells, succeeded by cone photoreceptors and RPE degeneration, eventually causing a gradual vision loss.¹⁰² The mechanism underlying RP involves a shortened rhodopsin due to mutations, which impair the cells’ capacity to fold and transport proteins, causing cell death via unfolded protein responses.¹⁰³ Clinical manifestations of RP include nyctalopia, restricted visual fields, and eventually central vision loss.^{104, 105} Genes such as Usher syndrome type IIA (USH2A, Usherin), retinitis pigmentosa 2 homolog (RP2), and phosphodiesterase 6B (PDE6B) harbor harmful mutations associated with RP.¹⁰⁶

RP is categorized into nonsyndromic and syndromic forms. The former exclusively causes retinal dystrophy without affecting other organs, with a prevalence of one in 5000 and is inherited as an autosomal-dominant (about 30−40% of cases), autosomal-recessive (50−60%), or X-linked (5−15%) traits.¹⁰⁷ The latter presents additional symptoms resulting from systemic disorders coexisting with retinal degeneration. Syndromic RP includes LCA, Usher syndrome, and Bardet–Biedl syndrome. LCA, with a prevalence of one in 80,000, is a RPE65 mutations-caused autosomal recessive hereditary disease featured by early vision loss, congenital pupillary reaction defects, and nystagmus noticed during infancy.^{108, 109} Usher syndrome, with a prevalence of about one in 10,000, presents as classic RP clinical symptoms along with varying degrees of hearing loss and vestibular dysfunction from USH gene mutations.¹¹⁰

Luxturna (voretigene neparvovec) represents a pioneering gene therapy with AAV2 vectors carrying a modified form of the human RPE65 gene, designed specifically for individuals with biallelic disease-causing variants in RPE65. In a phase III clinical trial involving 31 participants diagnosed with inherited retinal disease associated with biallelic RPE65 mutations, Luxturna administration yielded substantial improvements in visual function, with sustained benefits observed for up to 3–4 years. Notably, no severe adverse reactions were observed after 1 year (ClinicalTrials.gov Identifier: NCT00999609).^{4, 5, 111} Luxturna's success has spurred further investigation into gene abnormalities associated with hereditary retinal disorders, with numerous clinical trials ongoing and registered on clinicaltrials.gov. Technologies on efficient vector delivery, CRISPR/Cas9, and iPSCs-based cell transplants are accelerating personalized precision RP treatment.¹¹² CRISPR/Cas gene editing and gene regulation hold promise in improving the safety and efficacy of currently developed techniques, such as RNA editors. Although Luxturna exemplifies the potential of gene therapy, challenges remain in ensuring long-term safety, developing efficient delivery systems, managing immune responses, and addressing the high costs associated with gene therapy.

4.1.2 X-linked RP

RP GTPase regulator (RPGR) gene mutations commonly underlie the inherited X-linked retinal degeneration.¹¹³ Using sgRNA (Cas9) and AAV vectors, RPGR gene-editing has shown promise in preserving photoreceptors in mouse models with RPGR-related X-linked RP (XLRP).¹¹⁴ Subretinal injection of CRISPR–cas9 AAV vectors has successfully reinstated the open reading frame of RPGR^ORF15 in a subset of cells distributed widely in the retina of rd9 mice.¹¹⁵ A phase I/II clinical trial employing a dose-escalation design, a single sub-retinal injection of BIIB112, an AAV vector encoding codon-optimized human RPGR (AAV8-coRPGR), was conducted on individuals with XLRP resulting from RPGR mutation (ClinicalTrials.gov Identifier: NCT03116113), showing safety concerns primarily limited to steroid-responsive subretinal inflammation in high dose groups. Encouragingly, six patients exhibited improvements in the visual field, commencing at 1 month and sustained until the last follow-up assessment.¹¹⁶ A phase I/II dose escalation trial of rAAV2–RPGR for children and adults with XLRP due to faulty RPGR has been completed, with results pending publication (ClinicalTrials.gov Identifier: NCT03252847). Several other clinical studies are underway, including a phase I/II clinical trial assessing the safety and effectiveness of rAAV2tYF–GRK1–RPGR in participants with XLRP due to RPGR mutations (ClinicalTrials.gov Identifier: NCT03316560) and a phase III study assessing the safety and effectiveness of AAV5–hRKp.RPGR for treating Japanese XLRP patients with RPGR mutations (ClinicalTrials.gov Identifier: NCT05926583).

Gene therapy offers the possibility of reversing damage, recovering eyesight, and transforming the management of RP. As our understanding of the molecular mechanisms beneath RP improves, novel therapeutic avenues may emerge.

4.1.3 Age-related macular degeneration

Macular degeneration, often called AMD, is the primary contributor of severe, permanent vision loss in adults over 55.¹¹⁷ AMD manifests in two primary forms: dry AMD and neovascular AMD.¹¹⁸ Genetic factors are crucial players in AMD pathogenesis, with 103 AMD-related genes or loci identified thus far.¹¹⁹ Notably, changes in CFH and HTRA1 loci are significant contributors to AMD.^{120, 121} Additionally, genes related to lipid metabolism, such as ApoE,¹²² tissue inhibitor of metalloproteinases-3 (TIMP3),¹²³ and hepatic lipase,¹²⁴ have been implicated in the disease. Current clinical treatments for AMD include laser and radiotherapy,¹²⁵ photodynamic therapy with ranibizumab for polypoidal choroidal vasculopathy,¹²⁶ and anti-VEGF therapy, the primary therapeutic approach for neovascular AMD. The principal pharmacological agents utilized in anti-VEGF therapy include anti-VEGF monoclonal antibody fragments pegaptanib¹²⁷ and ranibizumab,¹²⁸ fusion VEGF binding proteins aflibercept¹²⁹ and conbercept,¹³⁰ and bevacizumab.

Gene therapy has demonstrated promise in AMD therapy. Research has indicated the long-term safety and effectiveness of AAV2 vector-mediated VEGF therapy.¹³¹ However, severe adverse events like atrial fibrillation, retinal detachment, and visual impairment in all doses, with incidence rates from 16.67 to 33.33% in different dose groups, happened in a phase I/II trial ascertaining the safety and tolerability of RGX-314, a rAAV vector containing coding sequences for soluble anti-VEGF after 24 weeks of injection (ClinicalTrials.gov Identifier: NCT03066258). Another phase I trial assessing the safety and tolerability of a single intravitreal administration of AAV2-sFLT01 revealed no severe eye-related adverse events (ClinicalTrials.gov Identifier: NCT01024998), suggesting that the tactic is safe and well tolerated across all dosage levels.¹³²

ADVM-022, developed by Adverum Biotechnologies, is presently undergoing a phase I clinical trial as an intravitreal gene therapy for neovascular AMD. Preliminary results have confirmed its safety and efficacy in maintaining sustained levels of aflibercept, suggesting a potential reduction in treatment burden and improvement in patient vision outcomes.¹³³ Suprachoroidal injections of RGX-314, an AAV8 vector expressing anti-VEGF-A Fab, have shown promising results in preclinical studies, suppressing VEGF-mediated vasodilation and vascular leakage in rats.¹³⁴ Another potential therapy with BT2, a dibenzoxazepinone, has exhibited inhibitory effects on vascular permeability and angiogenesis via suppressing retinal CD31, phospho-extracellular signal-regulated kinase, vascular cell adhesion molecule-1, and VEGF-A₁₆₅ expression in neovascular AMD.¹³⁵ Additionally, human complement factor I carried by AAV constructs is successfully expressed in the retina of C57BL/6J mice, demonstrating functional activity of the secreted proteins in vitreous humor.¹³⁶ HMR59, an AAV2 vector expressing sCD59, is presently under scrutiny in two phase I clinical trials: HMR-1002 (NCT03585556) for neovascular AMD (ClinicalTrials.gov Identifier: NCT03585556) and HMR-1001 (NCT03144999) for dry AMD (ClinicalTrials.gov Identifier: NCT03144999). Moreover, overexpression of AAV-mediated β-site amyloid precursor protein cleaving enzyme (BACE1) in the RPE has shown promising results, preventing retinal function loss and retinal degeneration for up to 6 months.¹³⁷ Two phase III clinical trials are ongoing to examine the safety of intravitreal injection of Zimura (Complement C5 Inhibitor) in geographic atrophy patients (ClinicalTrials.gov Identifier: NCT04435366 and NCT05536297).

In summary, expanding therapeutic targets beyond VEGF-A is an encouraging tactic for addressing the pathogenesis and clinical manifestation of neovascular AMD. Targeting various pathways may enhance treatment response, reduce resistance, and pave the way for future specialized therapies for neovascular and dry AMD.

4.1.4 Treatment of other ocular degeneration disorders

Stargardt disease is an autosomal recessive disease featured by macular degeneration and progressive visual impairment, primarily attributed to ABCA4 gene mutations.¹³⁸ A dual AAV vector split-intein adenine base-editing approach is able to rectify the most prevalent mutation in ABCA4 (c.5882G>A, p.G1961E) in human retinal organoid and mutation-carrying mice. This method sets the stage for accurate and effective gene editing in other neurodegenerative ocular diseases.¹³⁹ A phase II clinical trial initiated in 2022 is investigating vMCO-010 optogenetic therapy, an AAV2-based vector, for Stargardt disease (ClinicalTrials.gov Identifier: NCT05417126). In a previous phase I/II trial (ClinicalTrials.gov Identifier: NCT01469832), dose-dependent subretinal hyperpigmentation was observed in all patients post sub-retinal transplantation, persisting beyond systemic immunosuppression. Marginal, often transient, enhancements in best-corrected visual acuity (BCVA) were noted in four subjects. However, no overall benefit was detected at 12 months, with potential harm suggested in one high-dose case.¹⁴⁰

Enhanced S-cone syndrome is characterized by decreased rod photoreceptors, excessive proliferation of S-cones, and variable disruptions in M- and L-cone development. The transcription factor Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3), which governs ultimate differentiation and maturation of rod photoreceptors, is commonly implicated as a causative mutant gene.¹⁴¹

Mutations in the retinoschisis 1 (RS1) gene are connected to X-linked juvenile retinoschisis (XLRS), a degenerative retinopathy.¹⁴² A phase I/II clinical trial (ClinicalTrials.gov: NCT02317887) evaluating AAV8-RS1 gene therapy for XLRS has demonstrated that intravitreal administration of AAV8-RS1 results in systemic immune activation, evidenced by dose-dependent elevation of activated lymphocytes, macrophages, proinflammatory cytokines, and inflammation.^{143, 144} A phase I/II study assessing the safety and effectiveness of a rAAV expressing retinoschisis (rAAV2tYF-CB-hRS1) in 27 participants with XLRS revealed severe adverse events, including atrial fibrillation, cerebellar stroke, and pulmonary embolism in one out of six patients at the dose of 1 × 10¹¹, retinal detachment in one out eight participants at the dose of 3 × 10¹¹, retinal detachment in one out of 13 participants at the dose of 6 × 10¹¹. Other mild adverse events, including anterior chamber inflammation and iridocyclitis, were also observed in more than half of the participants (ClinicalTrials.gov Identifier: NCT02416622).

Bietti crystalline dystrophy (BCD) is an autosomal recessive inherited retinal disease characterized by chorioretinal degeneration. This condition is triggered by mutations in the cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene. BCD is distinguished by the appearance of yellow-white crystals, variable involvement of the cornea, and complex lipid deposits in the retina.¹⁴⁵ Several clinical trials are currently initiated, including a phase I/II trial evaluating the safety and effectiveness of ZVS101e (rAAV2tYF-CB-hRS1) administered by subretinal injection in BCD patients (ClinicalTrials.gov Identifier: NCT05832684), a multicenter study evaluating the safety and tolerability of a single subretinal application of VGR-R01, a novel AAV expressing human CYP4V2 (ClinicalTrials.gov Identifier: NCT05694598), and a phase I clinical study ascertaining the safety of rAAV2/8-hCYP4V2 gene replacement therapy administered as a single subretinal application in BCD patients (ClinicalTrials.gov Identifier: NCT04722107).

Choroideremia, a monogenic X-linked chorioretinal dystrophy, manifests as a progressive degeneration of RPE, choroid, and retina caused by mutations in the CHM gene, which encodes Rab escort protein 1 (RPE1), a ubiquitously expressed protein critical for Rab protein prenylation. REP1 is pivotal in intracellular vesicle trafficking, ensuring proper cellular transport mechanisms.¹⁴⁶ The first phase I/II clinical trial (ClinicalTrials.gov ref. NCT01461213) for CHM commenced in 2011, employing the AAV2/2 expressing REP1 in 14 CHM patients via subretinal administration.^{147, 148} A subsequent phase I/II clinical trial subretinally delivering AAV2-hCHM to the macula in patients with choroideremia revealed no differences in visual acuity between the injected and noninjected eyes at the 2-year postsurgery mark.¹⁴⁹ An ongoing phase I clinical trial is exploring the intravitreal application of an AAV capsid variant containing a transgene encoding a codon-optimized CHM gene (4D-110) (ClinicalTrials.gov ref. NCT04483440). Despite these efforts, gene therapy for choroideremia treatment still requires further preclinical investigations.

Leber hereditary optic neuropathy (LHON) is the primary cause of bilateral central vision loss among optic neuropathies..¹⁵⁰ This condition is primarily linked to major alterations in the mitochondrial genes ND1, ND4, and ND6, leading to increased oxidative stress within the optic nerve cells and subsequent nerve cell damage.¹⁵¹ A series of phase III clinical trials with ClinicalTrials.gov identifier numbers NCT02652780 (REVERSE), NCT02652767 (RESCUE), NCT03406104 (RESTORE), NCT03293524 (REFLECT), and NCT03295071 (REALITY) have been conducted to examine the effectiveness of a single intravitreal administration of GS010 to enhance retina functions and structures in individuals with LHON caused by G11778A mutation in ND4. The treated individuals exhibited a clinically relevant and prolonged progress in their visual acuity compared with natural progression of the disease.¹⁵² Another phase I trial assessed the safety of scAAV2-P1ND4v2 for LHON patients due to G11778A mutation in mitochondrial DNA (ClinicalTrials.gov Identifier: NCT02161380). The results indicated a beneficial safety and tolerability, with dosage-associated incidents of uveitis being the mere investigational product-associated adverse event. Some treated and fellow eyes in the chronic and acute bilateral groups exhibited betterment of ≥15-letter BCVA, whereas all study eyes (BCVA ≥ 20/40) in the unilateral acute group experienced a loss of ≥15 letters over 1 year despite treatment.¹⁵³ Overall, larger, randomized controlled trials are necessary to examine the safety and effectiveness of gene therapy for LHON.

4.2.1 Spinal muscular atrophy

SMA is a severe degenerative condition featured by progressive motor neuron deterioration.¹⁵⁴ This autosomal recessive disease is caused by a mutation in the SMN1 gene responsible for motor neuron survival and occurs in approximately one in 10,000 live births. SMA type I, the most severe form, affects approximately 60% of those diagnosed.¹⁵⁵ Several therapeutic approaches are available for SMA, with effective strategies focused on enhancing SMN production through modifying SMN2 splicing or faulty SMN1 gene replacement.^{156, 157} The most successful methods include viral gene therapy for defective SMN1 gene replacement and using antisense oligonucleotides or small molecules for SMN2 splicing manipulation, thereby increasing functional SMA protein production.¹⁵⁸

The compact nature of the SMN1 gene renders it amenable to delivery via AAVs, particularly AAV9, renowned for its capacity to penetrate the blood–brain barrier, thus serving as an optimal delivery vector. AVXS-101, a dsDNA, transports one or multiple copies of SMN1 to specific motor neurons’ nuclei, facilitating sustained gene expression.¹⁵⁹ A phase I clinical trial executed in 2017 examined the safety of intravenous administration of AAV9-SMN1 (AVXS-101) in infants with SMA type I. Three infants were given AVXS-101 at a lower dose and 12 received a higher dose as a single intravenous infusion. Although AAV-induced liver injury was observed in some cases, the treatment was generally well tolerated following prednisolone management. In the interim analysis conducted when the patients were between 20 and 32 months old, 11 infants in the higher dose group could sit unaided, nine could roll over, and two could walk independently.¹⁶⁰

These innovative therapies are expanding our comprehension of SMA's biology and pathogenesis, offering hope for disease reversal or reduction, especially with early intervention. By targeting different aspects of SMN deficiency, these treatments will provide valuable insights into SMN's roles, ultimately clarifying the disease's pathogenesis. Thus far, overexpression with this gene transfer approach has not shown harmful effects in preclinical models.

4.2.2 Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease triggered by DMD gene mutations, causing dystrophin protein loss.¹⁶¹ It manifests as muscle weakness, mobility loss by ages 9−14 years, and life-threatening cardio-respiratory complications.¹⁶² Due to its extensive size, the DMD gene is prone to spontaneous mutations, posing challenges for gene therapy. However, researchers are developing strategies to miniaturize the gene, inspired by a Becker muscular dystrophy patient who retained mobility despite a significant gene deletion. Current gene therapy trials by Sarepta Therapeutics, Pfizer, and Solid Biosciences are testing transgenes with varying spectrin repeats and hinges for safety and efficacy.⁸³ A nonrandomized controlled trial performed at Nationwide Children's Hospital in Columbus, Ohio assessed the safety and efficacy of intravenous administration of rAAVrh74.MHCK7.micro-dystrophin in DMD patients over 1 year. The findings indicate that this therapy may yield superior functional improvement compared with standard care¹⁶³ (ClinicalTrials.gov Identifier: NCT03375164). Delandistrogene moxeparvovec (SRP-9001) is a gene transfer vector expressing a truncated dystrophin protein. In a two-part, double-blind phase II study, this therapy was evaluated in aged 4–8-years-old DMD patients, with primary endpoints being changes in dystrophin expression and North Star Ambulatory Assessment score. Patients were randomized to administrate either a placebo or the therapy. The results demonstrated significant SRP-9001 dystrophin expression in all patients, with an average increase from the starting point to week 12 being 23.82% in Part 1 and 39.64% in Part 2. These findings suggest strong SRP-9001 dystrophin expression and NSAA stabilization after maximum 2 years of treatment¹⁶⁴ (ClinicalTrials.gov Identifier: NCT03769116). However, platelet transfusion and eculizumab treatment were necessary for a different participant with a condition resembling atypical hemolytic uremic syndrome caused thrombocytopenia. Pfizer has disclosed plans for a randomized, multicenter, double-blind, and placebo-controlled phase III trial involving 99 participants (C3391003) (ClinicalTrials.gov identifier: NCT04281485). Solid Biosciences launched the IGNITE DMD study, a phase I/II open-label clinical trial (ClinicalTrials.gov identifier: NCT03368742) using SGT-001, an AAV9 vector expressing SRs 16/17-encoded neuronal nitric oxide synthase (nNOS)-binding domain under the control of a CK8 muscle-specific promoter, aiming to improve perfusion in skeletal and cardiac muscles. Till now, six participants have been enrolled and treated, with three receiving a low dose (5.0 × 10¹³ vg/kg) and three receiving a high dose (2.0 × 10¹⁴ vg/kg). However, recurring issues with complement activation have led to two clinical holds by the US FDA.¹⁶⁵

Replacing the mutated DMD gene with a normal one theoretically holds the potential to cure the disease. However, the gene's large size and the muscle's widespread distribution pose significant challenges. To overcome these hurdles, researchers have developed a condensed micro-dystrophin gene and a systemic gene transfer method using AAV. Animal model investigations have shown improved muscle strength and reduced dystrophic cardiomyopathy. Several clinical trials aim to examine its safety and tolerability in DMD patients. Although these trials will not conclusively prove clinical efficacy, they will offer valuable insights into the potential of synthetic micro-dystrophin AAV vectors for whole-body DMD treatment.

4.2.3 X-linked myotubular myopathy

X-linked myotubular myopathy (XLMTM) is a genetic disease due to mutations in the myotubularin 1 (MTM1) gene located at Xq28.¹⁶⁶ MTM1 encodes myotubularin, a ubiquitously expressed phosphoinositide phosphatase critical for skeletal muscle development and maintenance.¹⁶⁷ Its loss-of-function mutations disrupt the excitation-coupling contraction mechanisms and T-tubule network organization, particularly evident in severe XLMTM, the most prevalent form presenting symptoms at birth. These symptoms include hypotonia, external ophthalmoplegia skeletal muscle weakness, and respiratory insufficiency.¹⁶⁸ A comprehensive international, prospective, longitudinal, natural history study involving 45 male participants aged 3.5 months to 56.8 years showed differentiation of three patient groups in motor function measure 32 total scores, grip and pinch strengths, and various respiratory measures. Some patients experienced motor milestone loss, whereas longitudinal data revealed a 2% decrease in motor function measured 32 total scores, indicating slow progression in male survivors regardless of phenotype. Additionally, 26% of patients exhibited detectable anti-AAV8 neutralizing activity.¹⁶⁶ In a study involving young patients with XLMTM, AT132 gene therapy showed promising outcomes. Treated patients demonstrated significant improvements in neuromuscular and respiratory functions, achieving key motor milestones and reducing ventilator dependency. Positive histopathological changes were observed in muscle biopsies. Despite some adverse events, the therapy's safety profile was manageable. However, following a patient's death, the US FDA suspended the study pending further investigation into the cause, emphasizing the critical importance of ongoing safety monitoring for AT132.¹⁶⁹ Although substantial advancement in systemic gene delivery has been made, challenges persist, necessitating thorough analysis and long-term surveillance of therapy effectiveness and safety. The severe complications observed in the XLMTM trial underscore the need for continued investigation and refinement of gene therapy approaches.

4.3.1 Alzheimer's disease

Alzheimer's disease is a neurodegenerative disorder pathologically defined as significant neuronal loss and the accumulation of intracellular neurofibrillary tangles and extracellular amyloid plaques in the brain.¹⁷² In early-onset, three specific genes have been identified as causative factors: amyloid precursor protein located on chromosome 21, presenilin 1 on chromosome 14, and presenilin 2 on chromosome 1.¹⁷² They harbor over 300 mutations, leading to elevated levels of overall β-amyloid (Aβ), increased Aβ42/40 ratios, and/or the formation of Aβ polymers.¹⁷² Studies have investigated the potential of nerve growth factor (NGF), a protein that could potentially restore and protect neuron functions in Alzheimer's disease. However, effectively delivering NGF has posed challenges. In an open-label clinical trial, gene transfer and stereotactic surgery were employed to administer NGF. Ten Alzheimer's disease patients received bilateral injections of a genetically engineered gene therapy vector (AAV2-NGF, CERE-110) into a specific brain region. The strategy was deemed safe and well tolerated over 2 years, without accelerated decline. Autopsies confirmed long-term, targeted NGF expression and activity, supporting the approach's feasibility and paving the way for a larger, double-blind, sham-operation-controlled trial.¹⁷³ Several other clinical trials are ongoing to address Alzheimer's disease, including a phase I trial examining the safety and tolerability of Libella gene therapy AAV-hTERT (ClinicalTrials.gov Identifier: NCT04133454), a phase I trial examining whether continuously delivering brain-derived neurotrophic factor (BDNF) into the brain by AAV2-BDNF can slow or prevent cell loss in individuals with Alzheimer's disease and mild cognitive impairment (ClinicalTrials.gov Identifier: NCT05040217), and a study evaluating the safety and potential toxicity of directly administering AAVrh.10hAPOE2 (LX1001) gene transfer vector expressing human apolipoprotein E2 (APOE2) into APOE4 homozygotes with Alzheimer's disease (ClinicalTrials.gov Identifier: NCT03634007). Despite initial setbacks, gene therapy is continuously investigated for treating Alzheimer's disease.

4.3.2 Parkinson's disease

Parkinson's disease is a neurodegenerative disease featured by slow movement, walking difficulties, and eventual cognitive decline due to dopamine-producing neuron loss in the basal ganglia of the brain. Gene therapy approaches have been explored to address Parkinson's disease. Preliminary clinical trials have explored gene therapy strategies using viruses to modify GABAergic neuronal signaling, such as AAV2-GAD (AAV-borne glutamic acid decarboxylase).¹⁷⁴ In a phase I trial involving the transfer of the AAV-GAD gene into the subthalamic nucleus, the procedure was deemed safe and well tolerated¹⁷⁵ (ClinicalTrials.gov Identifier: NCT00195143). Currently, Brain Neurotherapy Bio, Inc. is conducting a nonrandomized, open-label safety trial to explore the usage of AAV2-GDNF (AAV2-borne glial cell line-derived neurotrophic factor) for managing Parkinson's disease. In this phase I trial, AAV2-GDNF is delivered into the putamen. The trial is still in the participant recruitment stage, and no data have been published yet (ClinicalTrials.gov Identifier: NCT04167540). However, a phase II study of AAV2-neurturin (CERE-120) demonstrated no obvious difference in motor skill enhancement between the treatment and control groups. Serious side effects were noted in both groups, with a higher incidence in the AAV2-neurturin group. Additionally, a few patients in both groups developed tumors. Consequently, the study concluded that AAV2-neurturin treatment is not more effective than the placebo in improving motor skills over a year.¹⁷⁶ In gene therapy, precisely delivering target genes into a specific brain area using an appropriate vector is crucial. This process carries inherent risks, including disrupted walking patterns, damage to the dorsal root ganglia, loss of muscle coordination (ataxia), and elevated transaminase levels.¹⁷⁷

4.3.3 Canavan disease

Canavan disease is a type of leukodystrophy arising from harmful aspartoacylase (ASPA) gene mutations. ASPA, produced by oligodendrocytes, is crucial for breaking down N-acetylaspartate (NAA) via deacetylation. Increased NAA levels in the CNS can lead to various consequences, including abnormal myelination, parenchymal edema (swelling of brain tissue), and vacuolation (formation of small cavities in the white matter).¹⁷⁸ A phase I/II clinical trial is undergoing to assess the safety, tolerability, and pharmacodynamic activity of AAV9 BBP-812 in children affected by Canavan disease. AAV9 BBP-812 is a specially engineered vector carrying the ASPA gene, controlled by a universal promoter, aimed to reinstate ASPA expression in both neuronal and non-neuronal cell types (ClinicalTrials.gov Identifier: NCT04998396). A phase I/II clinical trial has been launched to examine the safety, pharmacodynamics, and effectiveness of a single intracerebroventricular dose of rAAV-Olig001-ASPA in up to 24 pediatric patients diagnosed with Canavan Disease. This trial represents the first-in-human protocol designed to evaluate the neurosurgical administration of a novel gene therapy vector targeting oligodendrocytes (ClinicalTrials.gov Identifier: NCT04833907).

4.3.4 Aromatic l-amino acid decarboxylase deficiency

Aromatic l-amino acid decarboxylase (AADC) deficiency disorder is a rare genetic condition stemming from specific variations in the dopa decarboxylase gene on chromosome 7, impacting neurotransmitter synthesis. Although symptom severity varies, approximately 80% of patients have a severe form, typically manifesting symptoms from infancy, including hypotonia, growth retardation, and significant motor impairments.¹⁷⁹ Chien et al.¹⁸⁰ conducted a trial assessing AAV2-hAADC gene therapy in patients with AADC deficiency, with 10 patients receiving the treatment. The results showed improvement in motor function and an increase in homovanillic acid concentrations, with one patient died from an unrelated cause. Reported adverse events were generally mild, with transient dyskinesia resolved with risperidone. This study highlights the possible effectiveness and tolerability of AAV2-hAADC gene therapy for AADC deficiency¹⁸⁰ (ClinicalTrials.gov Identifier: NCT01395641). In three separate clinical trials, children with AADC deficiency administrated eladocagene exuparvovec, an AAV2-mediated gene therapy, through bilateral intraputaminal infusions. The safety group consisted of 26 patients, whereas the treatment group included 21 patients. After treatment, average body weight increased, and the frequency of oculogyric crises improved. Although dyskinesia occurred as an adverse event, it is generally resolved with standard pharmacotherapy. Overall, eladocagene exuparvovec gene therapy showed positive effects on body weight, oculogyric crises, and dyskinesia in children with AADC deficiency.¹⁸¹ In another study involving 26 patients, eladocagene exuparvovec gene therapy showed significant and long-lasting improvements in motor and cognitive abilities for patients with AADC deficiency. Increased dopamine production, symptom relief, and improved quality of life were observed, with a favorable safety profile and manageable side effects¹⁸² (ClinicalTrials.gov Identifier: NCT01395641, NCT02926066). Looking ahead, research endeavors can explore noninvasive viral vector delivery or investigate alternative emerging treatments. Such studies hold promise for providing benefits to patients with AADC.

4.4.1 Hemophilia A

Individuals diagnosed with hemophilia A depend on exogenous administration of factor VIII to mitigate bleeding episodes in joints, soft tissues, and CNS. Although gene therapy is effective in treating hemophilia B patients, the extensive size of the factor VIII coding region has posed challenges for achieving comparable outcomes in individuals with hemophilia A through gene therapy. Rangarajan et al.¹⁸⁵ intravenously administered a single dose of a codon-optimized AAV5 vector encoding a B-domain-deleted human factor VIII (AAV5-hFVIII-SQ) in nine men with severe hemophilia A. Participants in the high-dose group achieved prolonged factor VIII activity > 50 IU/dL, leading to reduced bleeding events and elimination of the need for exogenous factor VIII. The main side effect was a slight augment in the serum alanine aminotransferase level. The only severe adverse event was preexisting chronic joint disease progression in one participant¹⁸⁵ (ClinicalTrials.gov Identifier: NCT02576795). In a longitudinal study, a cohort of 15 adults with severe hemophilia A (factor VIII level ≤1 IU/dL) who had previously administrated a single infusion of AAV5-hFVIII-SQ at different dosages were followed up for 3 years. The findings demonstrated that gene therapy utilizing the AAV5-hFVIII-SQ vector provided a durable and clinically significant improvement in individuals with hemophilia A¹⁸⁶ (ClinicalTrials.gov Identifier: NCT02576795). A phase III open-label, single-arm, multicenter trial assessed BMN 270, an AAV5 with a FVIII gene, in 134 patients with severe hemophilia A at a dose of 6 × 10¹³ vg/kg. This trial represented the largest gene therapy trial conducted on hemophiliac patients. The safety and efficacy results indicated that 132 individuals who were negative for the human immunodeficiency virus had mean FVIII activity of 41.9 IU/dL between 49 and 52 weeks. This was associated with a 98.6 and 83.8% decrease in treated bleeding and the use of FVIII concentrate, respectively. Transaminitis occurred in 85.8% of the subjects and was treated with temporary immunosuppression. No thromboembolic events, cancer, or use of FVIII inhibitors were reported. The initial observations appear promising¹⁸⁷ (ClinicalTrials.gov Identifier: NCT03370913). In a phase I/II trial, 18 men with hemophilia A were administered an AAV vector (SPK-8011) to promote factor VIII expression. Among the participants, 16 maintained stable factor VIII activity for more than 2 years, showing a remarkable 91.5% reduction in bleeding episodes. No significant safety issues were identified. The study has progressed to phase III evaluation, aiming to gather additional data on the efficacy and safety of this approach¹⁸⁸ (ClinicalTrials.gov Identifier: NCT03003533 and NCT03432520). In another phase I/II trial, B-domain-deleted FVIII codon-optimized cDNAs (BAY 2599023, AAVhu37.hFVIIIco) were delivered via an AAVhu37 capsid under the control of a liver-specific promoter/enhancer element. Nine individuals have been progressively included into one of the three dose cohorts (0.5 × 10¹³, 1 × 10¹³, and 2 × 10¹³ vg/kg) thus far. BAY 2599023 was delivered to six patients at 0.5, 1.0, and 2.0 × 10¹³ GC/kg dosages. All treated individuals showed symptoms of efficient blood coagulation and measurable, stable expression of endogenous Factor VIII (FVIII), demonstrating a successful proof-of-concept for the treatment¹⁸⁹ (ClinicalTrials.gov Identifier: NCT03588299).

4.4.2 Hemophilia B

Hemophilia B, a rare genetic disease linked to the X chromosome, is characterized by mutations in the F9 gene, responsible for producing blood coagulation factor IX, also known as the Christmas factor. Factor IX deficiency results in prolonged bleeding, which can occur spontaneously or following an injury. Hemophilia B predominantly affects males, with females mainly acting as carriers, resulting in a global incidence of about one in 30,000 male births.¹⁹⁰ In a clinical study, 10 patients with severe hemophilia B were systemic administrated varying dosages of scAAV2/8-LP1-hFIXco gene therapy drug via a single intravenous infusion. Their factor IX expression and activity were stable for over 8 years after the treatment, leading to a dramatic decrease in annual FIX concentrate usage and bleed rate. Although no late toxicities were observed, most patients had persistently high levels of anti-AAV8 capsid-specific antibodies. The study suggests that reducing the capsid load does not necessarily reduce hepatotoxicity in severe hemophilia B patients, indicating the involvement of other factors in this process¹⁹¹ (ClinicalTrials.gov Identifier: NCT00979238). In a phase III study, 54 men with hemophilia B received a single infusion of etranacogene dezaparvovec, regardless of any preexisting antibodies. The treatment was beneficial and safe for patients with predose AAV5 neutralizing antibody titers < 700, and no serious adverse events related to the treatment were reported. The use of factor IX concentrate also significantly decreased after treatment¹⁹² (ClinicalTrials.gov Identifier: NCT03569891). In a multicenter, open-label phase I/II clinical trial, investigators evaluated the safety and effectiveness of FLT180a in patients with severe or moderately severe hemophilia B, featured by factor IX levels ≤2% of the normal value. Although one patient with high factor IX level demonstrated severe arteriovenous fistula thrombosis, patients treated with low doses of FLT180a had normal factor IX levels over time when concurrently given immunosuppressant glucocorticoids, with or without tacrolimus¹⁹³ (ClinicalTrials.gov Identifier: NCT03369444, NCT03641703).

Ongoing research on modifications of AAV vectors to enhance transgene delivery and expression, along with initial successes in genome editing in animal models, highlight the need to continue developing “best in class” solutions for treating hemophilia B. The diverse AAV gene therapy trials present valuable alternatives for the hemophilia community, enabling the treatment of patients with existing antibodies against one serotype using alternative serotypes. Unlike hemophilia B, higher vector load are required to address hemophilia A. In addition, FVIII expression in subjects with hemophilia A seems to diminish gradually after treatment compared with patients with hemophilia B.

4.5.1 Heart failure

CUPID 2 trial, the largest gene transfer study on heart failure patients, concluded that AAV1/SERCA2a gene therapy did not improve primary outcomes compared with placebo, despite its safety. This outcome underscores the importance of further research into gene therapy for heart failure treatment¹⁹⁸ (ClinicalTrials.gov Identifier: NCT01643330). An analysis of explanted hearts after SERCA2a therapy demonstrated a diminished quantity of vector-derived DNA compared with preclinical studies, implying that inefficient delivery mechanisms could have played a role in the unsuccessful outcomes.¹⁹⁹ Based on these findings, the ongoing MUSIC-HFrEF1 trial is investigating whether a higher AAV dose could enhance vector uptake and improve efficacy. This phase I/II study includes a randomized, double-blind, placebo-controlled phase II segment involving 56 subjects with advanced heart failure (ClinicalTrials.gov Identifier: NCT04703842). In another phase II clinical trial (ClinicalTrials.gov Identifier: NCT00787059) in US medical centers (randomization occurred from July 19, 2010, to October 30, 2014), 56 individuals received either intracoronary administration of Ad5.hAC6 or placebo. The trial assessed primary outcomes such as duration and ejection fraction at different time points. Although no dramatic difference in exercise duration was noticed between the two groups, participants receiving the highest Ad5.hAC6 dose showed enhanced ejection fractions at 4 weeks but not at 12 weeks. AC6 gene transfer also boosted basal left ventricular peak but not arrhythmias. Notably, the hospital admission rate due to heart failure was lessened in the therapy group than the placebo group. The study concluded that AC6 gene transfer safely improved left ventricular function beyond standard therapy with a one-time application, suggesting larger trials are needed.²⁰⁰ The phase III randomized clinical trial FLOURISH, supported by Renova Therapeutics and involving 536 patients, further investigated this approach. However, in 2019, the trial was retracted from ClinicalTrials.gov to reassess the clinical plan and delivery strategy (ClinicalTrials.gov Identifier: NCT03360448). Currently, a phase I multicenter, open-label trial is ongoing to examine the safety, feasibility, and effectiveness of BNP116.sc-CMV.I1c in New York Heart Association Class III heart failure patients. Up to 12 subjects will receive a single intracoronary infusion, followed by 12 months of posttreatment follow-up and subsequent semi-structured telephone questionnaires every 6 months for 24 months (ClinicalTrials.gov Identifier: NCT04179643).

4.5.2 Coronary artery disease

Coronary artery disease stands as the foremost cause of mortality and the predominant cardiovascular a disease globally.²⁰¹ Gene therapy with members of the VEGF family emerges as a promising avenue for relieving symptoms in patients with refractory angina.²⁰² The therapeutic approach aims to stimulate new blood vessel formation and widen existing vessels by enhancing VEGF expression. This can be accomplished using genetically modified vectors, like VEGF-carrying adenoviruses. Upon introduction into the ischemic yet viable heart muscle, these vectors can trigger both angiogenesis and arteriogenesis.²⁰³ The phase I KAT301 trial was the inaugural exploration evaluating the safety and effectiveness of gene therapy using adenovirus-mediated VEGF-D^ΔNΔC (AdVEGF-D) delivered directly into the heart muscle of patients with refractory angina²⁰⁴ (ClinicalTrials.gov Identifier: NCT01002430). The trial revealed that regions treated with AdVEGF-D injections exhibited an enhanced blood flow reserve in the heart muscle, as measured by ¹⁵O-H₂O positron emission tomography. Furthermore, the treatment mitigated severe chest pain and improved overall quality of life and was deemed safe and well tolerated without severe associated side effects during the 1-year follow-up period²⁰⁵ (ClinicalTrials.gov Identifier: NCT01002430). Another clinical study examined the safety and preliminary effectiveness of encoberminogene rezmadenovec (XC001), a new therapy for refractory angina. In this phase I trial, patients received increasing doses of XC001 to assess its safety and initial effectiveness. The primary outcome was safety, monitored over 6 months. Efficacy was measured through changes in exercise duration, myocardial perfusion deficit, angina class, angina frequency, and quality of life. The findings indicated that the highest planned dose of XC001 was well tolerated and improved all parameters, warranting its advancement to phase II²⁰⁶ (ClinicalTrials.gov Identifier: NCT04125732).