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Signal Transduction and Targeted Therapy volume 9, Article number: 78 (2024 ) Cite this article scfv phage display
Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.
Gene therapy represents a groundbreaking approach for addressing genetic diseases, employing a range of strategies to modify gene expression within target cells using non-viral or viral vehicles.1 One common strategy is gene replacement therapy, where a functional copy of the faulty gene is introduced to living cells. A significant milestone for this strategy was reached in 2017 when the US Food and Drug Administration (FDA) approved the first gene therapy product, Luxturna, a gene replacement therapy for Leber congenital amaurosis type 2 (LCA2). Alternatively, gene silencing therapy aims to suppress or silence a target gene, mainly through RNA interference. For example, Patisiran, a small interfering RNA, is employed in the treatment of hereditary transthyretin amyloidosis.2 Another strategy is genome editing, which can be facilitated by clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to allow direct modifications of the somatic genome. Modified CRISPR systems that can perform RNA editing are also being developed. Moreover, gene expression may be altered epigenetically via DNA methylation, histone modification, and microRNA regulation. This method offers the advantages of being reversible and versatile, which allows it to be adaptable based on disease progression and responses to treatment. Innovative strategies such as leveraging suppressor tRNAs to enable readthrough of premature stop codons offer avenues to rescue pathologic nonsense mutations and restore gene function under endogenous regulation.3 Irrespective of the chosen strategy, gene therapy can be implemented either ex vivo or in vivo.4 Ex vivo gene therapy involves extracting patient cells, genetically modifying them, and reintroducing them back to the patients. In contrast, in vivo gene therapy directly delivers genetic materials to the target tissues. As of now, a total of 14 ex vivo and 29 in vivo gene therapies have obtained approval mainly through the FDA (Fig. 1). Of these approved gene therapies, 17 are non-viral-based, while 26 are viral-based. Various viral vectors have been studied for in vivo gene therapy, including adenovirus (Ad), retrovirus, lentivirus, and herpes simplex virus (HSV). Adeno-associated virus (AAV) vectors have emerged as the preferred choice in clinical trials and FDA-approved applications (Fig. 1). This is because of their broad tissue tropism, relatively good safety profile, and versatile manufacturing processes. Importantly, AAV is non-pathogenic, rarely integrates into the host genome, and can sustain long-term transgene expression. Moreover, vectors based on some AAV serotypes are inherently capable of efficient cellular entry and transgene expression, which enhances transduction efficacy.5
Approved gene therapy products and delivery platforms. Gene therapy products are generally developed for 1) Ex vivo gene therapy where affected patient cells are isolated, genetically modified in cell culture, expanded, enriched, and reinfused into patients to function as a living drug (e.g., CAR-T cells, Lyfgenia, Casgevy) and 2) In vivo gene therapy which is administered directly to patients to achieve therapeutic effects (e.g., Gendicine, Kynamaro, Imlygic, Luxturna, Onpattro). Delivery platforms for gene therapy drugs are primarily categorized into two groups: viral and non-viral-based. Viral-based gene therapy utilizes viruses as gene delivery vectors, including AAV, adenovirus, retrovirus, lentivirus, and herpes simplex virus. Non-viral-based gene therapy includes antisense oligonucleotides, siRNAs, and cell-based CRISPR genome editing. CAR-T chimeric antigen receptor (CAR) T cell therapy, SMA spinal muscular atrophy, AADC aromatic L-amino acid decarboxylase deficiency DMD Duchenne muscular dystrophy, LCA2 Leber congenital amaurosis type 2, hATTR hereditary transthyretin amyloidosis. * indicates non-FDA-approved gene therapy. Figure created with Biorender.com
Originally discovered as a contaminant in Ad preparations in the mid-1960s,6,7 wild-type AAV (wtAAV) is a non-enveloped virus containing a single-stranded DNA (ssDNA) genome with inverted terminal repeats (ITRs) at both ends (Fig. 2). Studies of AAV biology led to the successful cloning and sequencing of the wtAAV2 genome.8,9,10,11 In the 1990s and 2000s, diverse AAV serotypes with distinct tissue tropisms were identified, thereby introducing avenues for targeted gene delivery approaches.12,13 Studies reported that the wtAAV genome can be integrated into the host chromosome, facilitating stable, long-term transgene expression.14,15 During the 2000s, work was done to engineer AAV capsids to enhance tissue specificity and transduction efficacy as well as to improve the safety of recombinant AAV (rAAV).16 This has advanced to successful rAAV-based clinical trials, particularly for monogenic rare diseases, resulting in regulatory approvals of rAAV gene therapy products.17,18 In parallel, various methods to scale up rAAV manufacturing have been developed, leading to growing interest in rAAV-based gene therapy. More recently, the advent of CRISPR technology has revolutionized the landscape of gene therapy, enabling precise gene editing delivered into cells via rAAV.19
Historical milestones in AAV biology research and gene therapy development. Decades of studying AAV biology have led to crucial advances in understanding its structure, biology, vectorology, and gene therapy applications. Historical milestones in AAV research and development are summarized chronologically. These advancements paved the way for successful clinical trials and regulatory approvals for rAAV-based gene therapeutics to treat various human diseases. Figure created with Biorender.com
rAAV-based gene therapy has emerged as a potential cure for once-untreatable genetic diseases owing to its distinctive attributes, including its small size, non-pathogenic nature, versatile tissue tropism, episomally durable transgene expression, replication incompetence, engineerable capsids, and adaptability for diverse payload delivery.16,20 Despite some early successes, numerous concerns and challenges have restrained its widespread applications, particularly for complex disorders. These include limited cargo capacity, immune responses to high systemic doses, potential genotoxicity, challenges in achieving tissue specificity, and manufacturing complexity.16,20 Here, we describe AAV biology and engineering that is the foundation of rAAV gene therapy, discuss key principles of AAV vectorology and the current manufacturing methodologies, and provide overviews on clinical applications and challenges of rAAV gene therapy for treating a wide range of human diseases. Finally, we identify gaps in our current understanding, propose potential solutions to ongoing challenges, and outline future directions in this rapidly advancing field.
AAV has an icosahedral capsid composed of 60-mer subunits and a 4.7 kb ssDNA genome flanked by a 145 bp T-shaped ITR at each end. The capsid is assembled by viral proteins (VPs) VP1, VP2, and VP3 at a ratio of 1:1:10.21 VP3 is composed of conserved β-strands which are linked by surface-exposed variable loops. These structures shape the AAV surface morphology and determine AAV serotype-specific functions. The AAV capsid has a channel with a pore-like opening at each five-fold axis, protrusions surrounding the three-fold axis, and a depression at the two-fold axis.
The wtAAV genome contains two main open reading frames encoding four non-structural replication genes (Rep) and three structural capsid genes (Cap) along with an ORF for the assembly-activating protein, which is involved in capsid assembly.22 In addition, the function of membrane-associated accessory protein (MAAP), which is encoded on a different reading frame within the Cap gene, remains unclear.23 The ITRs largely serve as the viral origins of replication and provide the signal for packaging.24
Distinct serotypes of wtAAV are characterized by variations of VPs and ITRs and have been isolated primarily from humans and non-human primates (NHPs).12,13,25 Human seroprevalence studies have revealed that a significant proportion of populations possess neutralizing antibodies (NAbs) against multiple AAV serotypes. A study of 888 human serum samples from healthy volunteers found that NAbs against AAV2 were the most prevalent, followed by AAV1, AAV7, and AAV8.26 Another study showed that the most prevalent NAbs against serotypes other than AAV2 in serum from 552 human samples were AAV1 (74.9%), AAV6 (70.1%), AAV5 (63.9%), AAV8 (60.4%), and AAV9 (57.8%).27
As a Dependoparvovirus within the Parvoviridae family, wtAAV requires essential genes from a helper virus, such as Ad or HSV to facilitate the replication and transcription of its genome, including Ad E1, E2a, E4, and VA RNA genes.28,29 As a result, wtAAV cannot independently replicate and produce new viral particles but can establish a latent infection in host cells by persisting as episomal circular monomers or concatemers. Notably, in in vitro infections, approximately 0.1% of wtAAV2 genomes integrate into a specific region on the long arm of chromosome 19 (19q13-qter), termed the AAVS1 site.30 Although AAV is considered non-pathogenic, recent studies reported that AAV2 was connected with unexplained acute hepatitis in children worldwide.31,32,33 There is no direct evidence for the mechanism of how AAV2 triggers hepatitis, but it has been suggested that abnormal immune responses to AAV2 might mediate hepatoxicity.34 However, these studies acknowledged that COVID-19 infection may be a contributing factor and reported that most cases of acute hepatitis resolved without prolonged immunosuppression.
Naturally existing wtAAVs are rapidly evolving, generating a vast genomic diversity classified as viral “clades”. In the past two decades, at least 12 AAV serotypes and over 1000 variants have been identified from Ad stocks, human/NHP tissues, and other mammals or non-mammalian species.5,12,13,25 These serotypes have distinct preferences for various cells or tissues, which is known as tropism (Table 1).35,36,37,38,39 Genomic differences among AAV serotypes are primarily found in the variable regions of the virus capsid sequence, particularly VP3, which play a crucial role in determining the tropism. However, many other processes and interactions with host proteins may affect the tropism, including cell surface receptors, cellular intake, intracellular trafficking, nuclear import, viral uncoating, second-strand DNA synthesis, and genome circularization and concatemerization.
The wtAAV2 genome was initially cloned in the 1980s,8,9,11 serving as a template for rAAV. rAAV possesses an identical capsid sequence and structure to that of wtAAV, but lacks any wtAAV protein-coding sequences, instead incorporating therapeutic gene expression cassettes that are within 4.7 kb packaging capacity. The only viral elements in the rAAV genome are the ITRs.16 rAAV is primarily produced by trans-complementing Rep/Cap and Ad helper genes in transiently transfected HEK293 cells. rAAV genomes with AAV2 ITRs can be trans-encapsidated with capsids of different AAV serotypes to alter their transduction properties.
Despite an improved understanding of wtAAV biology, the mechanisms by which rAAV interacts with cellular surfaces and delivers transgenes into the nuclei of host cells remain poorly understood. A growing body of research suggests that the virus capsid initially adheres to the cell surface through primary receptors such as glycans, glycoconjugates, or sialic acid, followed by interactions with co-receptor proteins.40 For example, rAAV2 binds to heparan sulphate proteoglycan (HSPG), rAAV1, rAAV4, and rAAV5 primarily interact with sialic acid, and rAAV9 interacts with N-linked galactose.41,42 The distinct binding sites on the capsid are thought to determine its tropism, therefore efforts are being made around these sites to engineer AAV variants with enhanced transduction capabilities for specific cells or tissues.20 Recent genome-wide screening studies identified novel host proteins facilitating rAAV transduction, including the type I transmembrane protein KIAA0319L, which has been designated as the AAV receptor (AAVR).43 Another universal host protein, G protein-coupled receptor 108 (GPR108), plays a role in the transduction of several rAAV serotypes.39 Although knocking out AAVR and GPR108 in vivo reduces transduction, cell-surface binding is largely unaffected,39,44 suggesting that these host proteins primarily contribute to the transduction process post-attachment.
After binding to the cell surface, rAAV is internalized via endocytosis using different mechanisms, which can vary by cell types and rAAV serotypes.40,45,46 rAAV particles inside endosomes are subjected to pH-dependent conformational changes and then trafficked through the trans-Golgi network.47,48 The rAAV particles escape from the endosomes and trans-Golgi network and then enter the nucleus through the nuclear pore complex.49,50 Once the viral particles are inside the nucleus, the ssDNA genome is released and converted into double-stranded DNA (dsDNA) via a process known as second-strand synthesis. Transcription is then initiated from the self-primed ITR at the 3′ end of the genome.51 The genome can be made into a dsDNA structure by mutating an internal ITR motif, which allows for faster replication and enhanced transduction compared to the single-stranded rAAV (ssAAV) genome.52 However, this self-complementary AAV (scAAV) genome design reduces the packaging capacity by half.53,54 The dsDNA genome subsequently undergoes circularization and concatemerization, stabilizing the vector genome for episomal persistence in postmitotic cells.16 Notably, the ITR sequence can also serve as a recombinogenic element to facilitate vector genome recombination.16 The rAAV transduction pathway involves multiple cellular events and may fail or be destroyed by the host at any step, weakening or preventing transduction. Thus, a full understanding of this pathway will help identify additional key host factors affecting the transduction efficacy of rAAV.
Both the rAAV capsid sequence and the genomic DNA cargo, including promoters, transgenes, enhancers, and ITRs, are under intense investigation. This section introduces several engineering strategies to modify rAAV for better transduction efficiency and tissue specificity.
Capsid engineering is a principal strategy for developing rAAV tailored for different clinical applications. It mainly consists of three approaches: isolation of naturally occurring AAV variants, rational design, and directed evolution (Fig. 3). Recently, computer-based approaches using bioinformatic prediction and machine learning (ML) have emerged as novel tools to assist capsid engineering23,55 (Fig. 3).
Strategies applied for the development of novel AAV variants. a Natural occurring AAV variants can be isolated from human and NHP tissues using high-cycle PCR and high-throughput sequencing. b Rational design utilizes knowledge of AAV biology to modify the relevant amino acids in AAV capsid to enhance the transduction capability or evade immune surveillance. c Directed evolution is an engineering approach to develop novel AAV variants with designated specificity, including random or defined peptide insertion, capsid shuffling, error-prone PCR, and saturation mutagenesis. d In silico approach utilizes known capsid sequences to reconstruct ancestral AAV sequences. Machine learning is being used for predicting the relationship between specific sequences in the AAV genome with packaging capabilities and tissue tropism by using large datasets of samples transduced with AAVs whose genome had been mutagenized. Figure created with Biorender.com
Early in AAV development, human tissues were crucial sources for discovering new AAVs. From 1965 to 2004, only a limited number of AAVs were derived primarily from human clinical samples, which were subsequently vectorized and subjected to testing.5 AAV9 was first identified in human liver tissue, yet it has strong brain tropism due to its ability to cross the blood-brain barrier (BBB), making it a common choice for central nervous system (CNS) transduction.13 AAV7 and AAV8 were isolated from the heart and lymph nodes of rhesus monkeys and demonstrated superb tropism in skeletal muscle and liver.12
Studies indicate that 40–80% of people have antibodies against known wtAAV serotypes, highlighting the necessity of discovering more sero-distinctive AAVs.26,56 Screening for AAV serotypes in species other than humans and NHPs has been done to find capsids that can avoid the neutralizing effects of encountering pre-existing antibodies against human AAVs,57,58,59,60 but these may have limited transduction in humans. Thus, discovering human-derived AAV variants and screening for candidates with enhanced transduction and specific tissue tropism has been a common approach. AAVv66, an AAV2 variant isolated from a human sample by long-read sequencing, exhibits enhanced production and CNS transduction compared to AAV2.61 These findings indicate that natural variants can provide valuable tools for improved tissue-specific transduction. While only a few groups continue to evaluate natural capsid variants as potential vectors for gene therapy, rAAVs derived from natural AAVs remain the dominant choice in current clinical studies.
Rational design approaches attempt to make structural modifications in specific sites on rAAV capsids based on the understanding of rAAV structure and biology. Three key approaches have generally been employed: genetic mutation, insertion of nonviral domains to modify tissue affinity, and chemical modification.
Studies exploring the contributions of specific amino acid residues on rAAV transduction efficacy revealed that phosphorylation of surface tyrosine residues on rAAV2 capsids led to their degradation before nuclear entry.62,63,64,65 Modifying these residues to phenylalanine (Y444F/Y500F/Y730F) increased gene delivery in the CNS.66 While rAAV2 is not able to transduce photoreceptors through intravitreal injection, a variant containing substitutions of specific residues (Y272F/Y444F/Y500F/Y730F/T491V) achieved transduction of up to 25% of photoreceptor cells. Moreover, altering surface-exposed residues may enable tissue detargeting, thus improving the safety profile. For example, introducing H527Y and R533S substitutions, which were identified in samples from chimpanzee tissues, into the rAAV9 capsid resulted in reduced transduction in peripheral tissues after intravascular injection into both neonatal mice and a mouse model of a fatal pediatric leukodystrophy.67
Another rational design approach is to introduce functional domains into specific sites of the rAAV capsid. A study inserted a 15-mer binding domain of the human luteinizing hormone receptor (LH-R), resulting in the successful transduction in ovarian cancer cells in an HSPG-independent manner via the LH-R.68 By inserting cell-penetrating peptides into the rAAV9 capsid, a study identified two variants that were able to cross the BBB with improved CNS transduction after systemic delivery.69 The host immune response is a major barrier to rAAV being able to induce effective and long-term transgene expression. Structural studies have demonstrated that the NAbs recognition sites on rAAV capsids might be conserved.70 Engineered rAAV variants were able to evade NAbs in mice, NHPs, and human sera as determined by structural information from cryogenic electron microscopy (cryo-EM) images of rAAV1 capsids complexed with three murine monoclonal antibodies.70 Conjugating the rAAV capsid with biotin-polyethylene glycol (PEG)71 and N-acetylgalactosamine (GalNAc)72 may help evade NAbs, although this is potentially at the cost of altered tissue tropism and reduced transduction efficiency. One study used domain swapping to generate 27 chimeric Cap from rAAV2 and rAAV8 and demonstrated that the liver tropism achieved by rAAV8 was associated with the loop IV domain.73 Additionally, inserting arginine-glycine-aspartic acid (RGD) integrin binding motifs in AAVrh10’s variable region VIII improved cardiac-specific transduction and reduced liver distribution.74
Chemical modification without altering the amino acid composition of the rAAV capsid offers a promising approach in capsid engineering. A minor modification to the surface amino acids can alter the receptor binding affinity to affect transduction and tropism. A study used PEG-N-hydroxysuccinimide with an electrophilic succinimidyl propionic acid functional group to cross-link with lysine residues on rAAV2 capsid to enable NAb evasion.75 Glycated rAAV2 was found to have reduced binding to heparin and monoclonal antibody A20, resulting in remarkably improved transgene expression in muscles.76 One study covalently conjugated antibodies against skeletal muscle-specific protein CACNG1 to a variable region of rAAV9 capsid, leading to engineered capsids that can specifically express transgene in mouse myotubes with reduced liver targeting compared to unconjugated rAAV9.77 Likewise, another study engineered a hybrid capsid of rAAV9 and rAAVrh74 that can specifically bind to the skeletal muscle-enriched receptor integrin alpha V beta (AVB6). One resulting variant, LICA1, had significantly enhanced infectivity in human myotubes and the skeletal muscle of a mouse model of Duchenne Muscular Dystrophy (DMD).78 Alternatively, chemical modification of the capsid can be achieved through genetic code expansion, a versatile platform to incorporate non-canonical amino acids (ncAAs) with desired properties into proteins at a specific site in a gene of interest.79 A recent study using this approach to engineer a novel class of rAAV, termed Nε-AAVs, by inserting single ncAAs via engineered orthogonal prokaryotic tRNA/tRNA synthases.80 These mutant Nε-AAVs were successfully conjugated with functional molecules. In vivo studies demonstrated that the functional molecule conjugated to Nε-AAVs resulted in highly specific uptake in the target cells in xenograft animal models. Another study also showed that the incorporation of a ncAA at D374 in the AAV5 capsid led to enhanced transduction specifically in mouse lungs.81 Despite these advances, rational design approaches are limited by our incomplete understanding of rAAV structure and biology.
Surface panning of random peptides on an AAV capsid is another approach for directed evolution. For instance, a Cre recombination-based AAV targeted evolution (CREATE) system allows for attaching short-length peptides to rAAV9 capsid to engineer its properties.86 CREATE was used to generate AAV-PHP.B, an AAV9 variant that can cross the BBB to transduce the CNS in C57BL/6 mice.86 However, follow-up studies demonstrated that the properties of PHP.B capsid did not translate to other mouse strains or NHPs, suggesting a complex interplay between the engineered capsid and host factors which can differ between species.87,88
Subsequently, a novel rAAV evolution platform termed TRACER (tropism redirection of AAV by cell-type-specific expression of RNA) was developed. This platform is based on the recovery of viral RNAs expressed from a bulk capsid library carrying random peptide insertions in a cell-type-specific manner from animal tissue.89 A study in mice with peptide libraries displayed on rAAV9 capsids identified ten leading variants with up to 400-fold higher brain transduction over the parental rAAV9 following systemic administration. The same group recently reported that VCAP-102, a TRACER-engineered rAAV9 variant, displayed 20–90-fold increased transduction in diverse brain regions of NHPs compared to rAAV9.90 Iterative evolution of VCAP-102 led to capsid variants with 6–7-fold further improved BBB penetration. Similarly, TRACER-developed VCAP-100 derivatives showed up to 300-fold liver detargeting and six-fold higher brain transduction in NHPs over its parental AAV5.91
Directed evolution of rAAV capsids leveraging in vivo expression of transgene RNA (DELIVER) is another landmark method, which has led to the identification of highly functional muscle-tropic capsids in mice and NHPs.92 Cross-comparison of muscle-tropic variant capsid sequences in mice and NHPs identified a common RGD motif. Further analysis suggested that RGD-binding integrin heterodimers expressed in the target cells have a strong interaction with the viral variants containing the RGD motif. Researchers recently generated an RGD-embedded 7-mer peptide library inserted in variable region VIII of the capsid and identified variants with more than 20-fold enhancement in skeletal muscle transduction compared to the benchmark capsid.93
Directed evolution is a powerful tool to generate and identify variants with specific properties, but these properties are not always cross-species translatable in primates. For example, AAV-PHP.B showed enhanced CNS transduction in mice but not marmosets compared to AAV9, suggesting the necessity for cross-species evolution in capsid engineering.94 Nevertheless, a study sequentially evolving an rAAV capsid library across multiple species identified rAAV.cc47 as a potent cross-species variant with enhanced transduction capabilities over rAAV9 in the NHP brain and heart.95 7m8 is an AAV2 variant with a 7-mer insertion to strongly transduce the outer retinal layers such as photoreceptor and retinal pigment epithelium (RPE) cells through intravitreal injection mainly in mice.96 A phase II clinical trial (NCT04418427) using ADVM-022 (AAV.7m8-aflibercept) for treating diabetic macular edema (DME) was suspended due to severe adverse effects in several patients 16–36 weeks after receiving high doses (6.0 × 1011 vector genomes (vgs)/eye), including refractory intraocular pressure reductions, underscoring the translational challenges of engineered capsid in the field.
Computer-assisted rAAV engineering represents a cutting-edge approach that utilizes computational tools to enhance the design and optimization of rAAV. Studies have explored rAAV’s diverse properties from its evolutionary lineage and used reconstruction of the predicted ancestral genome to create novel capsid variants.55,97 Two similar studies computationally established the ancestral capsid library by rational design55 or adopting directed evolution.97 Both studies produced capsid variants with increased thermostability, an indicator of serotype identity, and promising biological properties desired for clinical application. Among these variants, Anc80L65,55 emerged as a promising vector for gene therapy for diseases associated with the inner ear98,99 or the eye.100
ML is extensively used in biomedical research, including medical image analysis, genetics, and drug discovery.101,102 A pioneering study applied ML to understand the rAAV2 capsid.23 They scrutinized the capsid’s fitness landscape, characterizing single-codon substitutions, and employing ML to design precise multi-mutated variants based on their impact on target tissues. Furthermore, deep learning has been used to diversify rAAV2 capsid variants, accurately predicting their viability.103 This approach reveals new possibilities for engineering the rAAV genome beyond the capsid, which has promising innovative applications in future rAAV development.
The rAAV genome consists of ITRs and a transgene expression cassette consisting of the gene of interest and regulatory components. Strategies to engineer the rAAV genome include modifying ITRs or introns as well as inserting tissue-specific promoters for transcriptional regulation and/or using an inducible expression system, codon optimization, and CpG motif reduction of the transgene cDNA, and microRNA (miRNA)-mediated post-transcriptional regulated retargeting.104,105,106,107 Each of these components has its own functions, and their individual and collective actions determine transgene expression, cell-type specificity, safety, and durability of rAAV transduction.
Traditional rAAV carries a single-stranded vector genome. Its transduction efficiency and rate depend on the second-strand synthesis of vector DNA before mRNA transcription occurs, which is the rate-limiting step. To increase transduction efficiency, strategies have been employed to mutate one of the ITRs, generating scAAV with a double-stranded vector genome.53,54 This allows scAAV to bypass the rate-limiting step of dsDNA synthesis, resulting in more rapid and efficient transduction of target cells.20,108,109,110 However, using scAAV has notable limitations: smaller packaging capacity (2.5 kb in scAAV vs 4.7 kb in ssAAV) and elevated risk of immune responses due to rapid accumulation of transgene products.111,112
Promoters in transgene expression cassettes are selected and optimized for specific needs such as tissue specificity, and expression level. The cytomegalovirus (CMV) or chicken beta-action (CBA) promoters are commonly used because they offer strong and ubiquitous expression, while tissue-specific promoters such as CK8 or MHCK7 provide targeted gene expression in muscle.92 The specificity of transgene expression within targeted tissues is crucial in order to achieve sufficient transduction efficiency with low dosing and minimize off-target effects. For example, a study employed a scAA9 vector carrying an endogenous human survival motor neuron 1 (SMN1) promoter to drive SMN1 expression specifically in neurons.115 This approach demonstrated a remarkable safety profile, notably decreasing liver toxicity, and enhanced therapeutic efficacy in the SMNdelta7 mice with spinal muscular atrophy (SMA). The outcome surpassed the performance of Zolgensma, an FDA-approved treatment utilizing the CMV/CBA promoter, thus underscoring the advantages of tissue-specific promoters. Additional regulatory elements such as upstream enhancers can be engineered to improve expression activity and specificity. Numerous enhancers have been designed in clinical gene therapy vectors with the goal of increasing transgene expression, thereby reducing the viral load.116 Another study reported that a fragment of the mouse methyl-CpG-binding protein-2 promoter enabled robust long-term expression specifically in neurons.117 ML-based multi-omics data allows to design of tissue-specific promoters, resulting in an over 1000-fold dynamic increase in transgene activity between on- and off-target tissues.118
Transcriptional regulation: The expression of the gene or protein of interest can be up or down-regulated to optimize and fine-tune the desired effect. The tetracycline-inducible system is a widely used strategy to achieve a specific level of expression, although it is unlikely to be translated to humans due to potential immunogenicity issues.119 Alternatively, a system was developed to use the immune suppressive drug rapamycin to activate responsive transcription factors delivered by two separate rAAVs.120 This strategy was subsequently optimized so that only a single rAAV is now required. This has been used to achieve inducible dose-response expression to rapamycin treatment for long-term expression of the desired transgene.121 Importantly, there was only minimal transgene expression without the administration of rapamycin, indicating that this could potentially provide a specific and safe regulatory system.
Post-transcriptional regulation: Another strategy is the use of riboswitches, which are non-coding RNAs that can bind specific metabolites and control gene expression.122 Riboswitches occur naturally, and the principles of ligand-mediated gene regulation are being exploited by engineering aptazymes for mammalian systems with improved regulatory ranges. Using riboswitches over other regulatory systems has the advantages of being compatible with FDA-approved drugs, the small size of the RNA, and little or no immunogenicity. Importantly, riboswitches have very short sequences, which is particularly important when combined with the limited packaging capacity of rAAVs, making it a powerful system for regulating rAAV gene therapy.123,124 Non-coding RNAs are highly versatile tools in transgene regulation, which is further highlighted by incorporating RNA aptamers with cleavable poly A signal (PAS) into the 5′-untranslated region (UTR) of the transgene. In the absence of a small molecule, PAS cleavage leads to mRNA degradation to silence transgene expression. The addition of the small molecule leads to transgene expression through maintaining the integrity of the mRNA, which can be combined with alternative splicing to more precisely control gene expression.125
Some regulatory systems require either exogenous addition or co-expression of certain elements to work correctly. The use of drug-inducible splicing as a mechanism of gene expression regulation has been pursued to mitigate this problem. This approach entails the delivery of a gene containing a premature stop codon to prevent the translation of the protein. The addition of a small molecule splicing inducer facilitates the inclusion of a specifically designed exon into the mature transcript and the exclusion of the stop codon, thereby enabling the translation of the protein of interest.126 The strength of the promoter and dose of the inducer molecule allows for the expression level of the target gene to be precisely activated. However, current technologies rely on intrinsic protein turnover mechanisms to reduce levels of the target protein.
Post-translational regulation: One strategy to modulate the half-life of a protein of interest is the use of a protein degrading system (degron). This allows for a reversible and rapid response to protein removal and recovery. Several of these systems have been established in vitro, but attempts to translate this to in vivo systems have found high levels of toxicity. However, optimizations have generated newer, promising systems for in vivo applications, such as incorporating a SMAsh tag on the protein of interest. A small molecule drug that has already been approved for human use is then introduced to prevent the detachment of the SMAsh tag, which leads to protein degradation.127 Combining inducible expression systems, alternative splicing strategies, and protein degradation approaches has the potential to allow for exquisite control of a target protein’s expression level.
One common method in expression cassette engineering involves codon optimization by using computer algorithms to identify rare or suboptimal codons in the transgene to be replaced with preferred ones. Studies have demonstrated significantly higher expression levels in codon-optimized transgenes such as FVIII and the human cystic fibrosis transmembrane conductance regulator (CFTR) in mouse models.128,129,130,131 Moreover, a codon-optimized transgene of aspartoacylase (ASPA), the causative gene of Canavan disease, remarkedly restored ASPA protein expression and rescued the lethal disease phenotype in a mouse model.132 While codon optimization can be an effective strategy for improving the expression of the transgene delivered by rAAV, such effects can vary depending on the specific transgenes and the host organism. Codon optimization methods might introduce unexpected immunogenicity and toxicity as well as impair other traits such as tropism. Indeed, a study found that codon-optimization introduced a large quantity of CpG motifs into a rAAV8-based FIX Padua gene therapy for patients with hemophilia B, which could lead to innate immune responses.133 A novel recurrent neural network-based tool was developed to optimize codon usage for specific cell types using data from mouse myocytes, neurons, and hepatocytes. This approach improved protein expression and reduced CpG dinucleotides, holding promise for enhancing tissue-specific gene therapy efficiency.134
Other cis-regulatory elements can be modified in the expression cassette to adjust gene expression and specificity. For example, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is a ~600 bp RNA element that is commonly added downstream of the transgene. The WPRE can enhance transgene expression both in vitro and in vivo by increasing the amount of mRNA transcribed independent of the transgene.135,136 Studies have cautiously reported potential oncogenic activity of the WPRE incorporated in lentivirus vectors delivered to mice.137,138 However, such risk can be reduced by removing the oncogenic sequence from the original WPRE.137
The inclusion of an intron in a transgene expression cassette can also improve gene expression in animals.139 This was supported by another study which showed that an intron increased transgene expression by 40–100-fold in vivo.140 However, the effects of introns on gene expression can be complex and depend on a variety of factors, such as the size and location of introns and the specific target cells. The selection of a proper polyA signal is important for optimizing transgene expression and stability (e.g., beta-globin, SV40, or bovine growth hormone (BGH)). A comparison study found that a modified shorter version of SV40 late polyA (135 bp) showed comparable transgene expression to a BGH polyA (223 bp) in a mouse brain.141 In addition, the inclusion of a Kozak sequence can further enhance the transgene expression.132
Ensuring confined transgene expression to target tissues is vital to avoid toxic overexpression and immunotoxicity. rAAV design incorporates cell-specific promoters as a primary strategy for targeted expression, and fine-tuning strategies are then developed such as adding miRNA binding sites in the 3′-UTR to inhibit expression in cells expressing the complementary miRNA. For example, adding miR-122 binding sites in rAAV9 was reported to enable CNS expression while retargeting liver, heart, and skeletal muscle.104 When rAAV carries a foreign transgene or one encoding a replacement gene in patients who have null mutations in the endogenous copy, the transgene product may be regarded as a non-self-antigen by antigen-presenting cells (APCs), which then process the transgene for major histocompatibility complex (MHC) presentation and immune clearance. One strategy to mitigate transgene immunity is to incorporate APC-specific miRNA binding sites, such as miR-142 and miR-652, into the expression cassette of rAAV to detarget transgene expression from APCs.142,143 The results showed a decrease in transgene-specific immune responses and sustained transgene expression in targeted cells in mice. Such a strategy has been translated in NHPs, in which broadly NAbs against human immunodeficiency virus were durably expressed after delivery by rAAV, thus highlighting this strategy’s potential for clinical translation.144
Another significant challenge in rAAV application is the delivery of transgenes that surpass its 4.7 kb packaging capacity. Examples include therapeutic transgenes like centrosomal protein (CEP290) for Leber’s congenital amaurosis type 10 (~7.5 kb),145 or CRISPR-based tools like cytosine base editor or prime editor 2 (>5 kb).146,147 Various strategies have emerged to overcome this hurdle by dividing a large transgene into two parts, with each encapsulated in an rAAV capsid. Co-introduction of these segmented transgenes into the same cell results in the reconstitution of the full-length transgene and protein. This reconstitution can occur at different stages within the genetic information flow.19 At the DNA level, inter-vector DNA recombination is facilitated by rAAV ITRs, a partial transgene sequence, or an optimized recombinogenic sequence present in both rAAV genomes.148,149,150 The overlapping sequence can be excised post-transcription through specifically designed splicing signals, resulting in a mature full-length mRNA that is subsequently translated into the desired protein. At the RNA level, trans-splicing between two transcripts from individual vectors, mediated by the splicing donor and acceptor present in each transcript, generates a full-length transcript and protein.151,152,153 At the protein level, protein reconstitution has been achieved through split inteins, the natural polypeptides capable of excising itself and ligating the nearby protein segments.154,155,156,157,158,159,160
The clinical applications of rAAV highlight the urgent demand for production and purification systems capable of producing substantial quantities and high-quality rAAVs to meet safety, efficacy, stability, and cost requirements (1.0 × 1015–1.0 × 1016 vgs per treatment).161 Because rAAV is unable to self-amplify by reinfection, its production requires the simultaneous expression of helper viral genes and rAAV-specific Rep and Cap genes for replication and packaging of the ITR-flanked vector genomes. Methods for rAAV production have been developed primarily with two strategies: transient transfection and viral infection. For transfection-based rAAV production, plasmid co-transfection into HEK293 cells is the most widely used production system, which was pioneered by Xiao et al. 29 rAAV production through viral infection involves the use of three different viruses, including AAV’s natural helper viruses, Ad and HSV, and baculovirus are used. rAAV production also involves either mammalian (e.g., HeLa, A549, and HEK293) or insect cells (e.g., Spodoptera frugiperda, Sf9) (Fig. 4).5 A newly developed tetracycline-enabled self-silencing Ad (TESSA) represents another infection-based system for large-scale, high-yield rAAV production in HEK293 cells.162 Each of these methods has its unique advantages and drawbacks in terms of flexibility, quality, and scalability, which will be discussed in more detail below. To purify rAAV produced by various methods, gradient sedimentation using either cesium chloride or iodixanol gradient ultracentrifugation is a commonly employed generic procedure for small-scale rAAV preparations.163,164,165 For large-scale vector production, and particularly for clinic-grade vectors that demand higher quality and quantity, affinity- and/or ion-exchange-based column chromatography is required.166 Finally, the development of stable packaging and producer cell lines is being actively pursued as the preferred next-generation rAAV production platform due to their straightforward scalability for large-scale production at a lower cost.
Current approaches to manufacture rAAV. Currently, two main platforms are used for rAAV manufacturing: transfection- and viral infection-based approaches. Plasmid transient transfection of HEK293 cells remains the most widely used method, while stable cell lines, baculovirus (BV) systems, and the HSV type I system offer scalable alternatives for large-scale production. The transfection-free helper virus system TESSA has been developed to produce high-yield rAAVs. Pharmaceutically inducible all-in-one producer cell lines may represent the next generation’s optimal manufacturing platform for rAAV-based drugs. BHK baby hamster kidney. Figure created with Biorender.com
Traditional rAAV production involves transfecting three plasmids carrying vector genome, AAV Rep/Cap genes, and Ad helper genes (VA RNA, E2A, and E4OEF6), typically at equimolar ratios, into HEK293 cells. Another essential Ad helper gene, E1a/b, is inherently expressed in HEK293 cells.167,168 This triple transfection protocol was subsequently optimized to a two-plasmid co-transfection system, involving one plasmid for the rAAV genome and another for Rep/Cap and Ad helper genes.169,170 Current methods can achieve approximately 80% cell transfection rate with peak virus yield at 48–72 h post-transfection.171
There are numerous challenges in viral vector manufacturing that lead to inconsistencies in quality, titers, and purity between batches,172 and the process is very labor-intensive.173 Recent research has focused on modifying the standard manufacturing process at the plasmid level to enhance both yield and purity with reduced cost. One notable recent study showed that low-cis triple transfection (1% or 10% cis plasmid) significantly reduced the transgene plasmid usage compared to the standard triple transfection (100% cis plasmid) and increased the in vivo transduction efficiency.174 Importantly, this method enables the packaging of yield-inhibiting transgene and reduces plasmid backbone contamination in both low-cis rAAV preparations and tissues of mice receiving this rAAV. Others developed a new all-in-one rAAV production system termed AAVone that combines Rep/Cap and Ad helper genes with the rAAV genome into a single plasmid. AAVone yields higher rAAV production compared to the triple transfection method, with lower batch-to-batch variations and reduced levels of replication-competent AAV (rcAAV). It also requires less plasmid DNA and eliminates the need for ratio optimization steps.175
The transient transfection approach presents flexibility by enabling the easy and rapid generation of rAAV with diverse transgenes and capsids. It can be scalable to an extent when using suspension cells, allowing for production to be increased simply by increasing the culture volume. In addition, the transient transfection method is flexible and versatile, and does not require time-consuming processes of establishing and maintaining stable producer cell lines for specific vector products. This facilitates a quicker turnaround time from vector design to production. Notably, this approach suffers from limited scalability and high cost.
The first rAAV gene therapy product to reach the market (uniQure’s Glybera,176 alipogene tiparvovec; now withdrawn from the market) actually utilized the Baculovirus Expression Vector Systems (BEVS), in which baculoviruses encoding Rep and Cap genes and carrying rAAV genome infect insect cells, such as Sf9, to produce rAAV.177 This system was originally optimized for large-scale protein production, but significant advancements have been made to accommodate for rAAV production. The first-generation BEVS involved splitting the Rep gene into two cassettes controlled by different promoters and modifying the Cap gene to include a non-canonical start codon (ACG instead of AUG). These genetically altered AAV viral sequences were incorporated into three separate baculovirus constructs, necessitating simultaneous superinfection of all three in a single cell.177 However, this method was initially successful for rAAV1 production but much less productive for other rAAV serotypes. Second-generation BEVS was more adaptable for additional rAAV serotypes and reduced the number of baculoviruses to two, one carrying Rep/Cap genes (Bac-CapRep) and another carrying the rAAV genome (Bac-Transgene).178 Subsequently, an attenuated Kozak sequence and leaky ribosome scanning were introduced to BEVS to ensure proper AAV gene expression, resulting in rAAV with significantly improved biological potency.179
Recently, BEVS has been further improved so that only one recombinant baculovirus is required for rAAV production. An engineered Sf9 cell line was created by integrating Rep/Cap genes, which then only needs one infection by a baculovirus carrying an ITR-flanked transgene, resulting in higher rAAV yields compared to the conventional three-Bac system.180 Another advancement introduced a highly regulated Rep gene control system to stabilize and allow appropriate Rep expression, leading to the production of potent and high-yield rAAV particles.181
One of the drawbacks of BEVS is the genetic instability of baculovirus. Baculoviruses naturally remove parts of their genomes during replication, which generates mutant viruses, some of which can become defective interfering particles.182,183 During the scaling-up process in cell culture, these mutants may outcompete baculoviruses with intact genomes, potentially reducing rAAV production. However, the genetic instability of baculoviruses can be potentially improved by either deleting specific genes or inserting transgenes into stable loci within the baculovirus genome.184,185 Additionally, BEVS is inherently not capable of assembling capsid proteins into AAV virions with the VP1, VP2, and VP3 at a ratio of 1:1:10, causing a reduction of transduction potency for the resulting rAAVs. A study demonstrated that the incorporation of artificial introns within the rAAV sequence, such as those using the baculoviral polyhedrin promoter, yielded infectious rAAV with high titers across various serotypes, reaching up to 1.0 × 1014 vgs/L.186 Furthermore, a study systematically compared rAAV produced by BEVS (Sf9-rAAV) to that produced by transient transfection (HEK-rAAV) using large-scale suspension cultures and found that Sf9-rAAV may be favored over HEK293-rAAV for its superior yields, full/empty ratio, scalability, and cost-effectiveness.187 However, further studies into the genetics and biology of baculovirus are necessary in order to harness the advantages of the BEVS platform (Fig. 4), such as its high scalability.
Stable cell lines for vector production offer notable advantages, including easy product characterization, scalability, and the capacity to generate higher yields with improved reproducibility and reduced cost.188,189 There are two types of stable cell lines for Ad infection-based rAAV production: packaging and producer cell lines. Packaging cell lines have stably integrated AAV Rep/Cap genes. A major challenge for rAAV production using packaging cell lines is that the cells need to be first infected with AAV’s natural helper virus, wild-type Ad, to initiate endogenous Rep/Cap expression, followed by secondary infection with Ad-AAV hybrid virus from which rAAV genomes will be rescued, replicated, and packaged.190,191 In contrast, producer cell lines have Rep/Cap and Ad helper genes, as well as rAAV genome, stably integrated into the host genomes. In this case, infection by wild-type Ad will activate Rep/Cap gene expression, facilitating the rescue, replication, and packaging of rAAV genomes.192 Stable cell lines derived from A549 and HEK293 cells have been adapted for suspension cultures for scalable rAAV production.193,194,195
Several HeLa-based cell lines, such as C12, H44, and B50, yielded high titers of infectious and rcAAV-free rAAVs through endogenous expression of Rep/Cap. An A549-derived stable cell line expressing Rep/Cap and a K209 cell line has also been reported to produce high yields of infectious rAAV per cell.193,196
There are several drawbacks to stable cell lines and Ad infection-based rAAV production systems. First, creating stable cell lines is often a complex, time-consuming, serotype-, and vector genome-specific process. Second, ensuring the characterization and stability of these cell lines can be challenging, with potential risks related to the impact of passage history on growth kinetics. Additionally, to ensure the safety of the final gene therapy product, robust downstream purification procedures are essential to eliminate any pathogenic Ad contaminants and oncogenic HeLa DNA.195
rHSV is another natural helper virus capable of providing helper functions for rAAV production.197,198 In order to produce rAAVs using an rHSV system, two distinct rHSVs carrying the AAV Rep/Cap genes and rAAV genome are created for sequential infections of HEK293 or baby hamster kidney cells, followed by downstream processing and purification. The first clinical trial using rAAV1 to treat α1-antitrypsin deficiency was produced using rHSV.199
The notable drawbacks of using the rHSV-based system for rAAV manufacturing include the generation of a sufficient quantity of rHSV particles and the specialized purification steps necessary to effectively eliminate neurotrophic and neurotoxic rHSVs and other associated contaminants from the final rAAV preparations.
Typical methods for rAAV production involve either helper-free plasmid transfection or the use of a helper virus. However, the helper-free system poses challenges when scaling up and the helper virus approach requires more robust downstream processing and purification to eliminate contaminating helper viruses and highly immunogenic VPs.195,200 A recent innovation introduced a helper Ad designed to inhibit its major late promoter (MLP), thereby restricting its replication and effectively overcoming these limitations.162 This was accomplished by introducing an inducible tetracycline repressor binding site into the MLP. This modified Ad functions normally when doxycycline is present but is restricted to genome amplification and early gene expression (known as the helper functions) without it. Through the utilization of this self-regulating Ad, researchers successfully facilitated the delivery of essential adenoviral helper functions, Rep/Cap genes, and the rAAV genome, resulting in a substantial enhancement in rAAV production with a nearly complete absence of contaminating Ad. Previously, numerous attempts to generate a genetically stable recombinant Ad expressing the AAV Rep gene in HEK293 cells have failed due to the cytotoxic and recombinogenic nature of Rep protein even at a very low level of leaky expression.201 However, the TESSA system tightly regulates the expression of the Rep gene, activating its expression only during rAAV genome rescue, replication, and packaging process, which is a major breakthrough. This system improved the production of various rAAV serotypes in both HEK293 and U87 cells, including rAAV2, rAAV6, rAAV8, and rAAV9.162 Moreover, this system can be scaled up to produce rAAV2 and rAAV6 with a significant improvement on vector yield over the transient transfection approach.202
Selecting which production system to use involves balancing flexibility, scalability, and quality, all of which play a vital role in the development of a safe and effective rAAV product to meet the demand, safety, and efficacy requirements for clinical applications.
To further improve the transient transfection approach, it is crucial to optimize multiple factors during both the production and purification processes, aiming for increased yield and reduced empty rAAVs. Minimizing empty vectors enhances vector potency, which allows for a lower injection dose to be used, thereby reducing immunotoxicity. Ongoing efforts are underway to optimize the upstream processes of rAAV production to improve rAAV yield. For example, adjusting the plasmid ratio by reducing the cis plasmid to 1% or 10% can achieve a yield of rAAV that is comparable to conventional triple transfection. This approach enables the packaging of previously unpackageable transgenes while also reducing backbone plasmid contamination and is proving to be a cost-effective strategy.174
A mechanistic modeling study using triple transfection has identified several key bottlenecks in rAAV production, including inefficient plasmid delivery to the nucleus and poorly coordinated timing of capsid synthesis and viral genome replication leading to the empty capsid.203 Strategies to address these issues include ensuring that viral DNA replication occurs before or in parallel with capsid production, such as early expression of Rep protein or late expression of Cap protein.
Efforts have also been made to identify small-molecule chemical additives that can boost rAAV production.204,205,206 A library of over 130 small molecules has been assembled to transiently antagonize a broad range of cellular innate antiviral pathways, effectively increasing viral production at scale.205 Imidazole-based small molecules have been shown to improve large-scale rAAV production through multiple signaling pathways.206
Modifying host factors can also affect production. Through a genome-wide screening strategy, studies identified several gain- or loss-of-function targets that can significantly affect rAAV production.207,208 Indeed, a systemic analysis of the cellular transcriptional response to transient plasmid transfection revealed that host cells actively sense rAAV production as an infectious insult and upregulate inflammatory and antiviral responses.209
To reduce empty AAV particles, a study created a hybrid Rep gene from different AAV serotypes with the 3′ end of the AAV2 Rep gene, resulting in a 2–4-fold increase in full capsids for non-AAV2 serotypes, suggesting that enhancing Rep function may improve rAAV production.210
Efforts in optimizing downstream purification processes have also led to improvements in removing empty capsids. The industry has transitioned from ultracentrifugation to column-based chromatography for effective purification of rAAV particles from impurities in large-scale production.211 Anion-exchange chromatography has become a major focus in the field, with modifications to various process parameters, including pH, various elution salts, excipients, surfactants, stabilizers, and osmolytes to enhance purification efficiency.212
Future manufacturing approaches should prioritize maximum flexibility, scalability, and quality while eliminating the need for plasmid transfection, virus infection, and chemical inducibility. Numerous efforts have been directed toward creating a stable and inducible producer cell line with these essential characteristics. One notable study exemplifies this approach by integrating all components necessary for rAAV production into HEK293 cells, including the rAAV genome, Rep/Cap genes, and helper coding sequences sophistically controlled by multiple inducible promoters. The resulting synthetic cell line generates infectious rAAVs upon induction.213 This all-in-one producer cell line approach allows for independent control over replication and packaging activities, ensuring high-quality rAAV production. Subsequent work has further optimized this producer cell line to enhance productivity by reducing the overexpression of the gene of interest, optimizing induction profiles, and mitigating proteasomal degradation of capsid protein by proteosome inhibitors.214 For large-scale vector production, another two-step platform for generating a stable and inducible producer cell line has been developed. Initially, an alpha cell line is created based on a proprietary CAP human cell line or HEK293 cells in suspension with stably integrated Rep and helper genes. This is followed by the integration of the Cap gene and the gene of interest, resulting in a polyclonal producer pool, followed by a single-cell cloning process, clone screening, and selection of the best-performing clone.215 rAAV production in this system is induced by doxycycline. In a proof-of-concept cell line with perfusion bioprocessing for rAAV8-GFP production, the rAAV per cell yield was enhanced 8-fold, with a significantly increased yield of full particles (30–40%) compared to the conventional batch process.
Enhancing vector genome integrity is another strategy to increase vector potency. A study using a long-read sequencing-based platform and bioinformatic pipeline identified different vector genomic populations from scAAV preparations.216 It concluded that robust DNA secondary structures inherent in vector genome designs can cause a significant truncation, impacting both production and vector efficacy. Another study reported the presence of truncated genomes and a unique genomic species that originated from a nickable ITR incorporating a small portion of payload and a chimeric sequence that joined to the plasmid backbone.217 This heterogeneity was also observed in a vector genome design carrying dual single-guide RNA expression cassettes in tail-to-tail configurations.218 Interestingly, the same group found notable differences in genome heterogeneity between human and insect cell-produced rAAV.219 These findings highlight the urgent need to scrutinize vector genome design and generation of heterogeneity for the efficient production of highly potent rAAV preparations, particularly for clinical vectors.
There have been remarkable developments in the clinical use of rAAV over the last few decades, underscoring its tremendous potential in gene therapy for a wide range of genetic and acquired diseases. Indeed, in a relatively short timeframe since the FDA approved Luxturna,220 there have been five additional rAAV gene therapy products introduced to the market (Fig. 1). Although rAAV-based therapies have shown impressive results in some clinical trials, numerous aspects require further investigation, including vector immunogenicity, dose optimization, and long-term safety. While meeting high-yield manufacturing challenges may not be an immediate issue for most monogenic diseases, producing rAAV at a large scale could become a bottleneck as more rAAV gene therapies are being developed for treating chronic prevalent human diseases. Furthermore, the regulatory framework for gene therapies is undergoing rapid changes. In this section, we will explore the latest developments in clinical applications of rAAV in major human diseases (Fig. 5), including ocular, neurological, metabolic, hematological, neuromuscular, cardiovascular, and oncogenic diseases. A table summarizing 238 clinical trials of rAAV-based gene therapies is presented in Supplementary Table 1.
Current clinical applications of rAAV in major human diseases. Clinical applications of rAAV across a spectrum of significant human diseases, including ocular, neurological, metabolic, hematological, neuromuscular, cardiovascular diseases, and oncology. Refer to Supplementary Table 1 for a comprehensive list of 238 clinical trials employing rAAV-based gene therapy for the aforementioned diseases. Figure created with Biorender.com
Ocular diseases are at the forefront of rAAV gene therapy for several reasons. The eye’s immune-privileged status reduces immune responses to rAAV. Its small volume necessitates low rAAV doses. Many ocular diseases are monogenic, making them suitable for gene therapy. Additionally, the relatively easy accessibility of the eye allows for various rAAV administration routes. Here, we highlight several rAAV-based ocular gene therapies, focusing on the treatment of monogenetic and acquired ocular diseases.
Voretigene neparvovec-rzyl (Luxturna) is a gene therapy to treat LCA2, a rare form of inherited retinal disease (IRD) caused by mutations in RPE65. This therapy delivers a functional copy of the RPE65 gene, correcting the deficiency in RPE cells, which are crucial for visual acuity. In a landmark phase III clinical trial, 29 LCA patients with confirmed biallelic RPE65 mutations were randomized to receive either Luxturna or no treatments as controls, followed by multi-luminance mobility testing, a measurement of visual function at specific light levels by requiring participants to navigate an obstacle course.221 The results indicate that Luxturna significantly improved functional vision (average of 1.8 light levels) compared to controls (average of 0.2 light levels) within one year. Moreover, full-field sensitivity threshold testing showed greater than 100-fold improvement in the intervention group by day 30, which was maintained over a year. Such beneficial improvements were sustained over 3–4 years in the subsequent follow-up studies.222,223 Due to the favorable results observed in this phase III trial, Luxturna was approved by the FDA in 2017 for the treatment of biallelic RPE65 deficient-associated retinal disease as the first gene therapy for genetic diseases.220
Given the heterogeneity of causative mutations in RP, various gene therapy strategies have been applied to this common form of IRD. Loss of MERTK, a crucial component in a signaling pathway that controls phagocytosis in the RPE, leads to photoreceptor degeneration and ultimately RP.224 This disease phenotype is autosomal recessive and accounts for ~3% of RP cases. A phase I clinical trial assessed subretinal injection of rAAV2-VMD2-hMERTK in six patients with MERTK-associated RP.224 While three patients showed vision improvement, sustained visual gains were only observed in one patient over 2 years, indicating that long-term investigation is needed.
Retinitis pigmentosa GTPase regulator (RPGR) gene mutation is a common cause of X-linked retinitis pigmentosa (XLRP) and primarily affects males. This is the most severe form of RP and is characterized by progressive vision loss due to dysfunction of photoreceptors and RPE cells.225 Three clinical trials have been conducted to assess the safety and efficacy of rAAV-based gene therapy to treat patients with RPGR mutations.226,227,228 Early results from one of the trials are encouraging, with some patients showing improvement in visual function and stability of retinal structure after receiving the treatment.226 A post hoc analysis of the gene therapy clinical trial (XIRIUS) and natural history study (XOLARIS) for XLRP revealed that the four participants who received the highest doses of cotoretigene toliparvovec (BIIB112/rAAV8-RPGR) had early improvements in retinal sensitivity and low-luminance visual acuity at month 12.229 In a phase II/III study, however, this gene therapy did not meet the primary endpoint of statistically significant improvement in light sensitivity in treated eyes.
Approximately 15% of RP cases are caused by autosomal dominant gain-of-function rhodopsin (RHO) mutations.230 Gene silencing or knock-out strategies can be used for this type of mutation. A preclinical study was conducted in NHPs using EDIT-103,231 an investigative gene therapy product containing two rAAV5 vectors to remove endogenous RHO through CRISPR/Cas9 and insert a codon-optimized RHO expression cassette that is resistant to targeted Cas9 cleavage to restore functional RHO expression. Early results have shown an encouraging improvement in RHO expression and visual function. However, its efficacy in humans is yet to be assessed.
In addition to gene replacement or editing approaches, other innovative strategies such as optogenetics have been investigated to treat RP independently of the genetic mutation. Optogenetics uses light to manipulate genetically modified cells with light-sensitive proteins. The initial assessment from a phase IIb clinical trial concluded that intravitreal injection of MCO-010, a multi-characteristic opsin encoded in rAAV2 designed to specifically target ON retinal bipolar cells, in patients with RP resulted in vision improvement with no severe ocular or systemic adverse events.232
AMD is the most common cause of blindness in developed countries. It is a progressive retinal condition featuring geographic atrophy and neovascularization at late stages. The mainstay of the treatment is to target vascular endothelial growth factor (VEGF), a major angiogenic factor involved in the pathogenesis of neovascularization. While anti-VEGF treatments have been proven to be efficient in alleviating disease progress and improving visual function in some patients, they require monthly injections and thus cause a high treatment burden while proving unsuccessful for a large proportion of patients. Gene therapy enables long-lasting, endogenous production of a therapeutic protein, solving many limitations of anti-VEGF agents, such as its short half-lives. As a result, there are an increasing number of clinical trials using rAAV to deliver genes for the treatment of both geographic atrophy and neovascular AMD.
GT005 (Gyroscope) is a rAAV2-based, one-time gene therapy product for geographic atrophy secondary to AMD that is delivered through subretinal injection. GT005 aims to mitigate retinal degeneration caused by an overreactive complement system in the aging retina by increasing the expression of the complement factor I (CFI) protein, a gene addition strategy to reduce inflammation and alleviate the degeneration of retinal cells. EXPLORE and HORIZON are phase II, multicenter, randomized, and controlled (treated vs. untreated patients) trials evaluating the safety and efficacy of GT005 in two different groups of patients with geographic atrophy (EXPLORE: patients with geographic atrophy secondary to AMD and with low CFI expression due to CFI variants; HORIZON: patients only with geographic atrophy secondary to AMD). Interim data indicate that the treatment achieves a sustained increase in vitreous CFI levels and a decrease in downstream proteins of CFI. The effect of the treatment is localized to the eye and no systemic increase in CFI levels has been observed. Nevertheless, the sponsor recently discontinued further development of GT005 because the futility criteria had been met.
sFlt-1 is a chimeric VEGF inhibitory protein consisting of domain 2 of Flt-1 (VEGF receptor-1) coupled to the human immunoglobulin G1 heavy chain Fc fragment.233 Gene therapy for neovascular AMD by using rAAV2 encoding sFlt-1 has been investigated through both intravitreal and subretinal administration.234,235,236,237 Intravitreal injection of rAAV2-sFlt-1 in a phase I clinical trial was reported to be safe and well-tolerated, while the expression level of sFlt-1 and the treatment efficacy was variable.238 Similarly, subretinal injection of the same vector was well-tolerated among the elderly.239 However, significant visual improvements were not observed, although this outcome was not the primary endpoint for the study.
RGX-314 (Regenxbio) is being developed as a subretinal treatment for neovascular AMD, consisting of rAAV8 encoding anti-VEGF monoclonal antibody fragments similar to ranibizumab. Initial assessment from a phase I clinical trial (ASCENT) showed that the treatment was safe and well-tolerated. The treatment outcome was dose-dependent, and several patients remained free of supplemental anti-VEGF injections for up to 18 months.
ADVM-022 (Adverum Biotechnologies) is a gene therapy product utilizing an engineered 7m8 vector derived from rAAV2 encoding aflibercept for the treatment of neovascular AMD. In a phase I clinical trial (OPTIC), a single intravitreal injection of ADVM-022 reduced the need for supplemental anti-VEGF injection in over 80% of patients for up to 92 weeks during the follow-up period.240
Current FDA-approved rAAV gene therapies for neurological disorders use two fundamentally different routes of delivery for localized versus widespread transgene delivery: stereotactic, anatomically confined rAAV delivery or widespread CNS transduction via intravenous (i.v.) delivery.
AADCD is an autosomal recessive disorder caused by mutations in the dopa decarboxylase gene, leading to AADC enzyme deficiency.241,242,243 AADC catalyzes the final step in serotonin and dopamine synthesis, with dopamine as a precursor for norepinephrine and epinephrine. This leads to a combined serotonin, dopamine, norepinephrine, and epinephrine deficiency.242
Patients present during the first months of life with muscular hypotonia, dystonia, oculogyric crisis, developmental delay, and autonomic dysfunction.241 A substantial delay between symptom onset (2.7 months) and diagnosis (3.5 years) has been reported.242 In its most severe form, patients miss all developmental milestones, lack head control, never learn to stand or sit, and die before the age of 10 years.241,244
Parkinson’s disease (PD) and AADCD have different etiologies (neuronal loss vs. enzyme deficiency) but result in striatal dopamine deficiency as a shared therapeutic target. This led to the rationale that one therapeutic vector could benefit both diseases. The first attempt to deliver intraluminal (the putamen is part of the striatum) rAAV expressing AADC (rAAV.AADC) was tested in 2008 in PD patients.245,246,247 Overall, the therapy was well-tolerated, with few procedure-related adverse effects, providing critical data on the safety and feasibility of stereotactic intracranial rAAV.AADC delivery was also associated with clinical improvement.246 Encouraged by the PD trials, this approach was extended to children with AADCD in 2012 in Taiwan.248 Four children, with the oldest being six years of age at the time of treatment, received bilateral intraputaminal infusion of rAAV2.AADC at a total dose of 1.6 × 1011 vgs. All patients showed improved motor scores and subjectively reported improved emotional stability. Interestingly, patients experienced transient post-infusion dyskinesia.248
A follow-up phase I/II trial in 10 patients used a slightly higher dose (1.81 × 1011 vgs), with the oldest patient being 8 years of age at the time of treatment, and no immunosuppression was used. All patients met the primary endpoints of clinical motor performance and cerebrospinal fluid (CSF) biochemistry improvement. Other measures, such as positron emission tomography (PET) imaging, showed significantly improved AADC activity. Again, post-infusion-related transient dyskinesia was noted.249
In 2019, a group in Japan treated six patients in a phase I/II trial with the same approach and a similar vector and dose (2 × 1011 vgs) as the Taiwanese group.250 The patient population, however, was more diverse regarding mutations and age at treatment, with the oldest patient being 19 years old. Similar to the 2017 Taiwanese study, patients experienced transient post-infusion uncoordinated movements of the mouth and extremities. Encouragingly, all patients improved in motor performance and oral food intake.
The anatomical target was selected based on PD pathology defined by dopaminergic neuron loss. However, in AADCD, dopamine-deficient dopaminergic neurons seem to persist. Targeting these dopaminergic neurons in the substantia nigra and ventral tegmental area to emulate physiologic dopamine synthesis was recently evaluated in a safety and efficacy trial.251 Seven patients between 4 to 9 years of age were assigned to two dosing cohorts (8.3 × 1011 and 2.6 × 1012 vgs total dose). All patients experienced improved motor function and mood. In 6/7 patients, oculogyric crises ceased completely. This clinical improvement was consistent with PET imaging results suggesting AADC enzyme activity. As in most trials, transient dyskinesia was observed post-infusion. Unfortunately, a direct comparison of both approaches is difficult at this stage and requires further studies.
Recently, long-term follow-up results from the Taiwanese trials reported significant improvement in motor function in 26 patients, with three patients able to walk.252 Interestingly, treatment age negatively correlated with motor scores, while dose did not.252 The most common treatment-emergent adverse effects (TEAE) were pyrexia and dyskinesia. Dyskinesia resolved in all patients but one. Notably, a positive correlation between dyskinesia severity/duration with treatment age was suggested.252 This correlation could be a critical observation to understand age-risk-benefit and might find equivalents in other CNS diseases. The therapy (Eladocagene Exuparvovec) was eventually licensed and has recently been approved by the European Medicines Agency for patients over 18 months of age (Upstaza).253
SMA is a model disease to targets widespread pathology in the brain and spinal cord by i.v. rAAV gene therapy. Over 3000 patients have been treated to date, providing critical insights into efficacy and safety. SMA is classified into variants I–IV.254 Variant I is the most severe form, with symptom onset in the first 6 months of life and death before the age of 2 years.255,256 It is characterized by weakness, motor development regression, and progresses to respiratory failure and death. SMA I is autosomal recessive and caused by loss-of-function mutations in the SMN1 gene.257 SMN2, a gene homolog to SMN1, produces a short version of SMN1. The number of SMN2 copies negatively correlates with the severity of the disease. The incidence varies based on country and ethnicity but is estimated as low as 1 in 7829.258
The first clinical trial results for SMA I were published in 2017.259 15 patients received either 6.7 × 1013 vgs/kg or 2 × 1014 vgs/kg of i.v. rAAV9 expressing SMN1 (rAAV.SMN1) under the control of the CB promoter with a CMV enhancer. The mean age was 6.3 months in the low dose and 3.4 months in the high dose cohort. The main findings were that patients had a reduced need for respiratory ventilator support and improved motor scores. 11/12 patients were able to sit unassisted for several seconds and gained the ability to speak. While the therapy was well-tolerated, elevated liver enzymes were common, but decreased with corticosteroid treatment.259
In two phase III follow-up studies in the US (STR1VE) and the EU (STR1VE-EU), an additional 22 and 33 patients, respectively, were enrolled and received 1.1 × 1014 vgs/kg i.v.260,261 The mean age at dosing was comparable in both studies (3.7 vs. 4.1 months). The US study had co-primary endpoints of unassisted sitting for at least 30 s and ventilator-free survival, while the EU study defined the primary endpoint as unassisted sitting for at least 10 s, and ventilator-free survival as the secondary endpoint. Both studies administered a prednisolone regimen. In STR1VE, 13/22 patients achieved independent sitting for at least 30 s, and in STR1VE-EU, 14/20 patients achieved sitting independently for at least 10 s, with one reaching 30 s. Of note, the EU trial had broader inclusion criteria, with some patients showing signs of more advanced disease, limiting the direct comparison of both studies. In addition, both trials reported elevated liver enzymes and thrombocytopenia after post-infusion.260,261
Long-term follow-up results from the initial START trial were reported in 2021.262 The longest follow-up was 6.2 years, and patients retained motor skills gained during the initial trial. Encouragingly, two patients acquired new motor milestones and could stand with assistance. The safety profile remained very good, with no TEAEs reported.262
The availability of an approved therapy allows the disease to be included in newborn screening, which could identify pre-symptomatic patients. This raises the possibility of prophylactic treatment.
The SPR1NT trial investigated the therapeutic outcome of prophylactic treatment with Onasemnogene abeparvovec (Zolgensma) at 1.1 × 1014 vgs/kg in 14 patients with two SMN2 copies (median age: 21 days) and 15 patients with three SMN2 copies (median age: 32 days).263,264 Prednisolone was given prophylactically and post-treatment. All patients with two SMN2 copies achieved the primary endpoint of independent sitting for over 30 s. 11/14 patients attained this milestone within the normal developmental window, with similar results observed for independent standing. With the understanding that there may be different definitions of walking, 9–10/14 children were able to walk independently, with 4–5 achieving this within the expected developmental window. Importantly, all patients remained free of permanent ventilation and alive at the end of the study. Elevated liver enzymes, cardiac serum markers, thrombocytopenia but no thrombotic microangiopathy (TMA), and transient areflexia/hyporeflexia were reported, which resolved in all but one patient and were classified as unrelated to the treatment.
Patients with three SMN2 copies manifest milder disease, which was reflected in independent standing for at least 3 s and walking for at least five steps in 14/15 children. 14/15 achieved standing and 11/15 achieved walking within the expected developmental window. Similar to the two SMN2 copy groups, all patients with three SMN2 copies remained alive and free from permanent ventilation at the end of the study. Reported TEAEs were comparable to the two SMN2 copies group.
The results in both cohorts emphasize the benefit of early pre-symptomatic treatment, which resulted in degrees of clinical improvement not observed before with Zolgensma. It also highlights the favorable safety profile of the treatment, although there have been reports of more severe hepatotoxicity and even death.265 Zolgensma obtained FDA approval in 2019, and as of March 20, 2023, over 3000 patients have been treated with Zolgensma, according to Novartis. Completed or ongoing studies are now exploring new aspects, including combination therapies or transitioning patients from conventional therapy to Zolgensma.266
Genetic defects causing metabolic disorders can have systemic or organ-specific manifestations, with implications on vector design and route of administration (e.g., lysosomal storage diseases with primary CNS manifestation). Here, Tay Sachs disease (TSD), Sandhoff disease (SD), and Canavan disease (CD) will serve as examples of neurometabolic conditions with currently active clinical trials (NCT04669535, NCT04798235, NCT04833907, and NCT04998396).
TSD and SD are lysosomal storage diseases, categorized as GM2 gangliosidoses resulting from autosomal recessive, loss-of-function mutations in the ubiquitously expressed gene encoding β-N-acetylhexosaminidase (HEX). HEX forms a dimer comprised of subunits α and β (HEXA), β and β (HEXB), or α and α (HEXS). TSD and SD are caused by mutations in the genes encoding the α- and β-subunits, respectively.267 Clinically, both diseases present in infantile, juvenile, and adult forms, which differ in onset and severity.268,269,270
One challenge is the need for a stochiometric balance of the α- and the β-subunits for optimal functionality of the HEX dimer. With rAAV’s limited packaging capacity, one strategy is simultaneous delivery of the α- and β-subunit with two separate vectors. Alternatively, a bicistronic rAAV has been developed to express the α- and β-subunits together.271,272
In 2022, an expanded access trial in two TSD patients demonstrated the safety of intrathecal (i.t.) infusion of two rAAV.rh8 vectors expressing α and β subunits. The patients were 30 months (1.0 × 1014 vgs; i.t.) and 7 months (4.05 × 1013 vgs; combined bilateral intrathalamic and i.t.) old and received immune suppression with rituximab, sirolimus, and prednisolone. The procedure and vector were well-tolerated. Although the functional outcome was not part of the study’s endpoints, both patients showed signs of disease stabilization. This first human TSD rAAV gene therapy study was a milestone in evaluating intrathalamic delivery and has paved the way for an ongoing clinical trial.273
Currently, two clinical trials are active in Canada (NCT04798235) and the US (NCT04669535). The Canadian phase I/II trial utilizes rAAV9 to deliver the HEXA and HEXB transgene with one vector for infantile TSD or SD patients via a single i.t. dose. In contrast, the trial in the US is designed as a dose escalation study of four different vector doses administered via bilateral intrathalamic route and i.t./cisterna magna combined. The study uses two rAAV.rh8 vectors delivering the HEXA or HEXB transgene. Results from these trials are pending.
CD is an autosomal recessive inherited progressive neurodegenerative disease hallmarked by spongiform degeneration and elevation of N-acetylaspartate (NAA). Loss of function mutations in the enzyme ASPA causes accumulation of NAA in the CNS and urine. Affected children typically never learn to walk, talk, control the movement of their head, or gain any level of independence.274,275 Many patients succumb to the disease within the first years of life. A clinical trial evaluated the safety of intracranial rAAV administration for the first time in the treatment of CD.276 After this clinical trial, studies to optimize vector design could achieve varying degrees of age-dependent prevention and reversal of the disease in mice.132,277,278,279,280 It is likely that differences in vector design and route of administration might account for the variable outcomes reported in preclinical studies.
Combining intracerebroventricular (i.c.v.) and i.v. routes of administration were employed to reduce the overall vector dose in an early single-patient trial.281 However, comparative studies are needed to evaluate this strategy.
Currently, there are two clinical trials ongoing, with one actively enrolling at the time of writing (NCT04833907 and NCT04998396). The two trials use different vector designs and routes of administration (intracranial delivery vs. i.v. infusion). This difference in route of administration necessitates differences in dose. Data for both trials have been presented at scientific conferences and look to be promising. With both trials ongoing, direct comparison is not feasible at the time of this writing.
Non-oncologic hematologic conditions can be classified into cellular or acellular diseases, meaning that genetic mutations affect cellular function (e.g., sickle cell disease)282 or components of blood (e.g., hemophilia),283 respectively. The distinction is crucial for gene therapy as it determines the target cell/organ.
In cellular hematologic conditions, the target is a cellular blood component, typically bone marrow. Blood cells have a short half-life, so directly targeting them by rAAV loses therapeutic efficacy quickly as the cells turn over. In contrast, bone marrow stem cells constantly divide to produce new cells, which dilutes the rAAV genome. Nevertheless, this may be a desirable effect if the host genome can be edited or if the gene therapy-expressing cells gain a competitive advantage. Acellular components, in contrast, are released by cells into the bloodstream. This provides an opportunity to express the transgene in any cell type that can export proteins into the bloodstream.
Hemophilia A and B are prime examples of hematologic disorders caused by a deficiency in secreted acellular blood components.283 The estimated prevalence for hemophilia A and B are ~12:100,000 and 4:100,000, respectively.284 Hemophilia A and B are caused by factor VIII (FVIII) and factor XI (FXI) deficiency, respectively, and both are recessive X-linked conditions.285 FVIII is synthesized in endothelial cells, whereas FIX is produced in hepatocytes.286,287,288
Hemophilia A and B patients have a substantially increased risk of bleeding spontaneously or with minor trauma, causing high morbidity and mortality (e.g., intracranial hemorrhage).289,290 In addition, repeated bleeding causes local reactions and disabling cellular degeneration (e.g., hemophilic arthropathy).291 Notably, patients have reduced overall survival compared to the general population.290
Prior to gene therapy, treatment of severe hemophilia A or B required repeated administration of the deficient factor or drug to modulate the coagulation cascade.292,293,294 Healthcare spending for those drugs has been rising, reaching ~$1.5 billion in the US in 2019.295 Gene therapy emerges as an ideal modality due to its potentially enduring effect and the clinical knowledge (i.e., natural history) in diagnosing and treating hemophilia, a critical aspect in gene therapy development that is often missing in ultra-rare conditions. In addition, FVIII and FIX are secreted, which means ectopic expression in a tissue could produce the factor (e.g., muscle). Hemophilia severity is classified based on plasma FVIII or FIX activity: severe <1%, moderate 1–5%, and normal 5–40%.285 This provides clear target levels to achieve partial (>1%) or complete (>5%) disease correction. Such meaningful thresholds rarely exist or are known in other diseases.
First attempts to develop gene therapy for hemophilia using viral delivery platforms predate the successes of rAAV.296,297 These early non-rAAV studies could not achieve lasting therapeutic factor levels or trigger immune responses against the viral vector.
In 1997, two studies could demonstrate persistent human FIX (hFIX) expression using rAAV (rAAV.hFIX) via intramuscular (i.m.) or intraportal vein (liver-directed) injection.298,299
A possible challenge could arise from FIX activity inhibiting antibodies, which were found after i.m. rAAV.hFIX.300 However, using species-specific transgenes might ameliorate the formation of such antibodies or other immune responses, as demonstrated in hemophilia B dog studies that utilized canine FIX (cFIX).301,302 While intraportal delivery is invasive and dogs required FIX transfusion prior to the procedure, slightly higher cFIX plasma levels were observed versus the i.m. study.301,302 However, this conclusion is unreliable due to differences in study parameters, including vector dose.
While the change to the rAAV8 capsid and codon-optimization of the transgene showed promising improvements, it did not consistently achieve therapeutic FIX activity. To overcome these challenges, a newly engineered capsid with increased liver tropism and a codon-optimized Padua FIX variant was designed.309 Notably, the dose (5.0 × 1011 vgs/kg) was 4-fold lower than the high dose in the 2011 and 2014 trials (2.0 × 1012 vgs/kg). Despite the reduced dose, the FIX activity reached a mean of 33.7% (range 14–81%).309,310 Prophylactic immunosuppression was not administered and only two patients required prednisolone for transient liver enzyme elevation, highlighting the excellent safety profile. Clinically, the annualized bleeding rate decreased significantly from a mean rate of 11.1 to 0.4.310 Overall, the increased activity of the Padua variant turned out to be highly beneficial and was utilized in subsequent clinical trials.
The hemophilia B trials uncovered several aspects of rAAV gene therapy in humans that were not anticipated, including a gradual decrease in transgene expression and immune responses. Currently, FDA-approved gene therapy for hemophilia B (i.v. rAAV5 Padua, Hemgenix, Etranacogene dezaparvovec) is on the market, while other rAAV products (verbrinacogene setparvovec (FLT180a) using an rAAVS3 capsid and Fidanacogene elaparvovec, formerly known as SPK-9001 and PF-06838435) are in clinical trials but await FDA approval.
A notable challenge for hemophilia A is the ~7 kb FVIII transgene, exceeding rAAV’s packaging capacity311 and necessitating the use of a truncated form. The therapeutic efficacy of a truncated FVIII was evaluated in nine patients by i.v. rAAV5 delivering a codon-optimized FVIII variant (FVIIISQ; missing the B-domain).312 The results from different dosing groups (6.0 × 1012 vs. 2.0 × 1013 vs. 6.0 × 1013 vgs/kg) demonstrated that the highest dose achieved therapeutic FVIII activity for the 52-week study period. Encouragingly, only the high-dose patients received immune suppression, and no clinically adverse effects were identified.312 However, transgene expression decreased over time.313
A separate trial evaluated a rAAV3-derived engineered capsid expressing FVIIISQ at four doses ranging from 5.0 × 1011 to 2.0 × 1012 vgs/kg in 18 patients.314 While it achieved therapeutic benefit, two of the 18 patients lost transgene expression entirely due to an anti-rAAV capsid immune response despite using glucocorticoids. Of note, attempts to wean several patients off glucocorticoids resulted in a cellular immune response. Although vector properties between the trials by Savita et al. 312 and George et al. 314 might explain the difference in transgene expression and maintenance, it was hypothesized that high expression levels might trigger an unfolded protein response with subsequent FVIII loss.314
A follow-up phase III trial using rAAV5hFVIIISQ enrolled 134 patients receiving a single 6.0 × 1013 vgs/kg i.v. dose.315 This one-time treatment significantly increased FVIII activity and reduced exogenous FVIII use, and the annual bleeding events improved compared to exogenous FVIII prophylaxis. Elevated liver enzymes were found at a median of 8 weeks, which decreased with glucocorticoid treatment in 96.2% of the patients.315 The two-year follow-up data showed a similar trend as in the previous phase I/II trial, with decreased annual bleeding rate and exogenous FVIII use. However, decreasing transgene expression over time was observed as well. Unexpectedly, the FVIII levels in the phase III trial were lower than what was found in the phase I/II trial at one- and two-year follow-up.316 This therapeutic vector recently obtained FDA approval (roctavian, Valoctocogene Roxaparvove, rAAV5hFVIIISQ), and it will be vital to conduct follow-up clinical studies to determine its durability.
Neuromuscular diseases comprise conditions affecting neurons and muscle cells,317 with muscular dystrophies primarily affecting muscle cells. DMD is the most common dystrophy,318 and the FDA recently granted approval for an rAAV-based gene therapy.319 DMD is a rare X-linked and progressive disease with a prevalence of ~15.9–19.5/100,000,320 which primarily affects males, although rare cases in females have been reported.321 It is caused by mutations in the dystrophin gene resulting in absent or dysfunctional dystrophin protein. It manifests with muscle weakness, loss of the ability to walk, respiratory impairment, and cardiomyopathy,322,323,324 with the latter two commonly being the cause of patient mortality.
The dystrophin gene consists of eight promoters and 79 exons to generate a ~11.4 kb cDNA and 427 kDa muscle isoform protein.321 Mutations typically lead to a truncated protein in DMD patients.321 The large dystrophin cDNA size poses a major challenge to rAAV-based gene replacement therapy because it exceeds the packaging capacity, so strategies have focused on identifying the minimally required protein sequences. Two different dystrophin transgenes have been developed: ~6–8 kb mini-dystrophin and ~4 kb microdystrophin.325,326,327,328 The mini-dystrophin needs to be divided and delivered by two rAAVs, while the microdystrophin fits into one rAAV.325 Other strategies using rAAV involve exon skipping,329 GALGT2 delivery330 (shown to prevent muscle pathology in mice), and gene editing to correct the dystrophin gene or activate non-muscle dystrophin isoforms using CRISPR.331,332 The focus here is on strategies that have been translated to human application.
In 2006, a trial evaluated a hybrid rAAV2.5 expressing miniature dystrophin by i.m. injection in six patients, aged 5–11 years.333 Patients were divided into two dosing groups: 2 × 1010 and 1.0 × 1011 vgs/kg. While some muscle fibers were found to be mini-dystrophin-positive in 2/6 patients at day 42 post-treatment, the failure to find dystrophin-positive cells at later time points might point towards an immune-mediated process. This is also supported by findings of transgene-specific T cells. Surprisingly, 2/6 patients were positive for T cells against a domain of the mini-dystrophin gene prior to receiving gene therapy, suggesting that the mutated endogenous dystrophin might not be sufficient to induce immune tolerance.333
To achieve broad targeting of affected muscle cells throughout the body, a trial evaluated the safety and therapeutic efficacy of i.v. infusion of 2.0 × 1014 vgs/kg rAAV.rh74.micro-dystrophin in four patients with a mean age of 4.8 years.334 Encouragingly, all patients in this phase I/II trial improved clinically and histopathologically, which correlated with age and disease severity at the time of treatment. In contrast to the earlier trial results from 2010, T-cell activity against the micro-dystrophin protein was not detected. Liver enzyme levels rose after rAAV infusion but remained moderate and normalized with corticosteroid treatment. No signs of TMA were found.334 A randomized, double-blinded, placebo-controlled clinical trial (NCT03769116, delandistrogene moxeparvovec) involving 41 patients was just completed. Notably, this therapy obtained FDA approval in 2023.319
An alternative strategy to gene replacement therapy involves the use of disease-modifying genes. In 2022, a first-in-human trial treated two patients (6.9 and 8.9 years of age) with rAAV.rh47 expressing GALGT2 at 5.0 × 1013 and 2.5 × 1013 vgs/kg per leg via bilateral femoral vein isolated limb perfusion, respectively.335 Isolated limb perfusion has been widely evaluated in animals as an attempt to increase muscle transduction but also reduce the total dose. GALGT2 acts as a β1,4 N-acetylgalactosaminyltransferase in muscle glycosylating α-dystroglycan and has shown positive effects on muscular pathology. No relevant liver toxicity was reported, despite the detection of antibodies against the rAAV capsid and mild T-cell activity against rAAV and the GALGT2 protein. While the patient number was too small to draw significant conclusions, it was noted that the younger patients receiving the higher dose showed greater improvement.335
Other ongoing trials are still active but not recruiting, including a randomized phase I/II trial by Solid Bioscience (NCT03368742) and a non-randomized phase Ib trial by Pfizer (NCT03362502), which both utilize rAAV9 to express truncated dystrophin under the control of muscle-specific promoters. These trials evaluate increasing i.v. doses and include children and adolescents but exclude patients with advanced degrees of cardiac and respiratory impairment.323
Defining eligibility criteria can be particularly challenging and highlights current gaps in understanding and predicting the interaction between rAAV gene therapy and the disease. A recent single-patient DMD trial using i.v. rAAV9 at 1.0 × 1014 vgs/kg expressing dCas9 linked to VP64 to activate expression of cortical dystrophin ended fatally.336 The patient, who was non-ambulatory and had advanced disease at the time of dosing, developed worsening cardiac function on day five and acute respiratory distress syndrome on day six post-dosing, resulting in cardiopulmonary arrest and death on day eight post-treatment. The concluding assessment led to the hypothesis that the patient developed cytokine-mediated capillary leak syndrome, as tests for the humeral or cellular immune response against the rAAV capsid or transgene were negative. There were also no signs of TMA, as has been seen in high-dose DMD trials. A second patient, who was part of the Pfizer trial (NCT03362502), died six days after receiving 2.0 × 1014 vgs/kg of rAAV9. The patient was younger and received double the dose as in the single-patient trial above. Although no autopsy was performed, the current hypothesis centers on an innate immune response in the cardiac muscle causing heart failure (HF).337
HF is a syndrome defined by structural or functional alterations of ventricular filling or emptying.338 It is highly prevalent with ~6 million people affected in the US.339 Calcium is crucial in cardiac function and is also a target for conventional drug development. One preclinically successful strategy is the overexpression of ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2 (SERCA2a), which pumps calcium into the sarcoplasmic reticulum (SR) during the cardiac relaxation process.340,341
The CUPID trial evaluated overexpressing SERCA2a using rAAV1 (rAAV1.SERCA2a).342,343 It was designed as a phase I/II trial, with the phase I portion being an open-label sequential dose escalation study342 and phase II being randomized, double-blind, and placebo-controlled.343 During the phase I study, a single intracoronary administration for heart-directed rAAV1.SERAC2a delivery was evaluated at 1.4 × 1011, 6.0 × 1011, and 3.0 × 1013 vgs (n = 3 patients per cohort). All treated patients had New York Heart Association (NYHA) Class III HF at the time of treatment. Some patients displayed symptomatic improvement and tolerated the treatment well. One patient died, which was determined to be unrelated to the treatment. Overall, the favorable safety profile encouraged the transition to the phase II portion of the study, which enrolled 25 patients receiving treatment and 14 receiving placebos. Doses were assigned to three cohorts, 6.0 × 1011 (n = 8), 3.0 × 1012 (n = 8), and 1.0 × 1013 vgs (n = 9).343 All patients had NYHA Class III HF at the beginning of the phase II study. Overall, a trend of improvement in the treated patients versus placebo was observed; however, most outcome criteria did not reach statistical significance, except for left ventricular end-diastolic volume. The study confirmed the favorable safety profile observed in phase I.343
The three-year follow-up results of the CUPID phase II could not demonstrate significant improvement in survival. However, a cumulative analysis for non-terminal events reached statistical significance encouraging CUPID 2.344 CUPID 2 assigned a total of 250 patients to the treatment (n = 123) or the placebo group (n = 127).345 Similar to prior trials, critical outcome measures did not show significant differences (e.g., survival). Interestingly, early terminal events were significantly increased in the intervention group.345
Other studies investigated the benefit of rAAV1.SERCA2a on left ventricular end-diastolic volume (AGENT-HF) or safety in patients with concomitant left ventricular assist device (LVAD) (SERCA-LVAD) and the impact of circulating NAbs.346,347 Both trials were terminated after discouraging CUPID 2 trial results emerged.
Although these trials did not result in clinically significant improvement in outcome, they highlight the potential of rAAV gene therapy for cardiac diseases. New, promising cardiac gene therapies using rAAV are being developed, including using rAAV9 to express LAMP2B for the treatment of Danon disease, an X-linked disorder causing severe cardiomyopathy. A phase I (NCT03882437) trial has completed recruitment, and the interim results suggest a favorable safety profile and clinical improvement, with occurrences of TMA and acute kidney injury being resolved by treatment.348,349 A corresponding phase II (NCT06092034) study is actively enrolling males of at least 8 years of age as a single-arm study for administration of a single i.v. infusion. The trial is expected to be completed in 2025.
rAAV has shown promise in preclinical studies for cancer, but its translation to use in humans has been limited.350,351,352,353 At the time of this writing, only one trial using rAAV for ex vivo gene therapy in gastric cancer is active (NCT02496273). Thus, this section focuses on preclinical achievements and rAAV-based strategies to treat oncologic conditions. For a more detailed understanding, we refer readers to several in-depth reviews.354,355
Anti-tumor strategies using rAAV share similar targets and concepts as conventional anti-tumor regimens. However, gene therapy offers specific advantages, such as modulation or knock-down of gene expression. rAAV transduces dividing and non-dividing cells, enabling broad cell targeting within heterogeneous tumor tissue. This property can be used to deliver cytokine transgenes to tumor cells to recruit immune cells and trigger an anti-tumor immune response.
A proof-of-concept study showed that rAAV-mediated interferon β (IFNβ) expression suppressed colorectal cancer cells in a xenograft mouse model.356 In a non-randomized, non-blinded, retrospective human study, the addition of IFNβ to temozolomide therapy extended survival in glioblastoma (GBM) patients.357 Driving expression of IFNβ by rAAV-transduced (rAAV.IFNβ) tumor cells can overcome IFNβ’s short half-life and boost therapeutic efficacy, as evidenced in an invasive orthotopic xenograft GBM mouse model, where intracranial delivery almost doubled overall survival.358
Taking advantage of rAAV’s adaptable capsid, tumor-directed capsid engineering can increase transduction efficiency and specificity while mitigating possible adverse effects. Indeed, rAAV displaying a Her2 ligand showed increased transduction specificity.359 The expression of HSV thymidine kinase converts ganciclovir into a toxic product which is subsequently incorporated into the tumor DNA to induce cell death. Delivery of this gene reduced tumor burden and extended survival compared to an animal group that received the approved anti-cancer drug Herceptin.360
While engineering capsids to deliver cancer therapeutics offers high specificity, this approach is time-consuming and labor-intensive. An alternative approach is to screen existing rAAV capsid libraries for tropism after delivery by different routes of administration to determine the best possible combination. For example, rAAV7 and nine have been found to achieve high transduction of prostate tissue in mice.361 This enhanced transduction of prostate tissue can be used to knock down miRNAs associated with prostate cancer, which has been found to slow disease progression in a mouse prostate cancer model.362,363
These proof-of-concept studies highlight promising anti-cancer strategies using rAAV. However, critical questions must be addressed before human application is feasible. There are concerns regarding the safety of sustained therapeutic transgene expression, particularly after tumor elimination or relapse. Similar to conventional chemotherapy, recurrent tumors might be resistant to or able to escape the therapy, making ongoing transgene expression ineffective. Given that cytokine therapies often have substantial side effects, unregulated transgene expression could cause additional complications. Improved regulated gene therapy strategies need to be developed together with advances in understanding of tumor biology.
rAAV capsid performance is tightly connected to the route of administration, prompting exploration of different delivery routes, including intravascular, direct intra-tissue injection, and delivery into pre-existing body cavities or fluid space.364,365,366,367,368,369,370,371 Each route and capsid selection must be determined in the context of the disease, target area, organ system, and other factors such as patient age (Table 2).
human egfr antibody Another challenge is the translation from preclinical to clinical studies. To mitigate vector costs and risks from high doses as used in i.v. delivery, intra-CSF space administration is widely utilized for widespread CNS transduction.373,374,375 The challenge of extrapolating findings in mouse, rat, or even larger animal brains to humans is daunting. For example, rAAV9 can achieve global brain transduction in mice, where the distance between the lateral ventricle and cortex is merely a few millime