Basic and Clinical Application of Adeno-Associated Virus

Abstract

Traditional gene therapy (gene replacement) has made a breakthrough in treating inherited diseases. Adeno-associated virus (AAV) has emerged as a highly promising vector with innate ability, boosting the development of gene replacement and gene targeting. With the recent advance of engineered nucleases that work efficiently in human cells, AAV mediated-genome editing with nucleases has raised hopes for in situ gene therapy of inherited and non-inherited diseases. Here, the applications of AAV-mediated genome editing are highlighted, and the prospect of AAV and nucleases that will render extension of such success in clinical gene therapy is discussed.

Introduction

Due to the detailed exploration of the human genome in recent decades, hundreds of causative genes of human genetic disorders have now been identified. This has been greatly facilitated by the rapid progress in next-generation sequencing technology, which has allowed the identification of disease-associated genes for many inherited diseases, especially monogenic diseases.¹ Traditional clinical therapeutic approaches such as surgery or small molecule-based therapeutics cannot effectively treat these diseases. Consequently, gene therapy has emerged as a revolutionary concept, as it can potentially directly alter and repair the pathogenic gene. To date, a series of adeno-associated virus (AAV)-based gene therapy clinical trials have been performed for monogenic conditions, including lipoprotein lipase deficiency,² hemophilia,³ and choroideremia.⁴ In addition, AAV-mediated gene therapy holds promise for the treatment of complex disorders, such as heart disease⁵ and age-related macular degeneration (AMD).⁶ More details of clinical trials involving AAV can be found online (ClinicalTrials.gov). Particularly, AAV-RPE65 has been approved as a drug (LUXTURNA™) to treat Leber congenital amaurosis (LCA), which is a milestone in AAV-mediated gene therapy.^*

AAV is considered a very promising gene-delivery vector, and has been tested in >100 clinical trials carried out over the last 15 years. Notably, with the ability to engineer AAV vectors and the development of genome editing enzymes, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeat (CRISPR)-Cas9, innovative genome editing methods are being developed to treat inherited and non-inherited diseases. AAV can serve as a donor template for homologous recombination (HR) with remarkably high efficiency. Double-strand breaks (DSBs) triggered by nucleases is also an important factor that greatly boosts the efficiency of HR-mediated genome editing.⁷

AAV's replication relies on a helper virus such as adenovirus or herpesvirus. Therefore, it is considered to be a safe vector in gene therapy. Compared to other viral or non-viral vectors, recombinant AAV (rAAV) genomes are maintained predominantly as episomal circular forms and possess sustained expression, low pathogenicity (immune response), versatile serotypes (tissue specificity), and highly infectious efficiency, resulting in widespread potential applications for both basic and clinical research (Table 1). In a clinical trial for the treatment of hemophilia B (NCT00979238), AAV8 vectors with an F9 transgene were well expressed and tolerated in patients without the detection of toxicity after 3 years of vector administration.³ Consequently, by using AAV for the delivery of genome editing nucleases, new genome editing strategies will be developed with great potential to generate disease models and correct mutations underlying genetic disorders.

Table 1.

Comparison of different viral and non-viral vectors

Vectors	Advantages	Disadvantages
Retrovirus	High efficiency of transduction; long-term stable expression	Random integration; gene silencing; proto-oncogene activation; unable to infect nondividing cells
Lentivirus	Infection of both dividing and nondividing cells; long-term stable expression	Random integration; gene silencing; proto-oncogene activation
Adenovirus	Infection of both dividing and nondividing cells; large capacity; no integration into host genome	Lethal to people with immunodeficiency; high immune response, presence of neutralizing antibodies
Adeno-associated virus	Infection of both dividing and nondividing cells; low random integration; long-term stable expression; low immunogenicity; variable tissue tropism	Low capacity; presence of neutralizing antibodies
Non-viral vectors	Large capacity; low immunogenicity; most of them are easy to synthesize	Low transfection efficiency; transient expression

AAV Structure and Serotypes

AAV is a non-enveloped, replication-defective, single-stranded DNA virus, which only replicates in the presence of a helper virus. It was discovered in 1965 as a contaminant during the preparation of a simian adenovirus, and classified into the Parvoviridae family due to its small diameter of approximately 22 nm (Fig. 1).⁸ AAV is composed of three structural proteins (VP1, VP2, and VP3) and a linear, single-stranded DNA (ssDNA) genome of approximately 4.7 kb, which contains 145 nucleotides inverted terminal repeats (ITRs). The first 125 nucleotides of the ITRs are palindromic and of great importance for viral replication. Because of alternative splicing and the presence of multiple start codons, AAV cassettes have two open reading frames (ORFs): rep and cap. Rep encodes four overlapping proteins (Rep78/Rep68 and Rep50/Rep42), which are responsible for replication, virion assembly, and site-specific DNA integration, as well as translational regulation. Cap encodes three structural proteins (VP1, VP2, and VP3) and a viral assembly factor (assembly-activating protein [AAP]), which contribute to the icosahedral capsid with the coordination of AAP (Fig. 1).⁹

Figure 1.

The structure of wild-type adeno-associated virus. Color images are available online.

Because of the diversity of its serotypes, which target various cell-surface receptors, AAV can infect different cell types with variable tissue tropism. Up to now, 12 different serotypes have been well characterized (AAV1–AAV12), and >100 new serotypes have been discovered more recently. The discovery of tissue tropism provides the basis for the selection of tissue-selective AAV vectors for targeted gene therapy (Table 2).

Table 2.

Tissue tropism of various AAV serotypes

Target tissue	AAV serotypes	Refs
Brain	AAV1, AAV2, AAV5, AAV9, AAV-PHP.B	83 –86
Blood	AAV1, AAV6, AAV8, AAVHSC	261, 28, 34, 87, 88
Cardiac	AAV2, AAV8, AAV9	5, 89, 90
Liver	AAV2, AAV6, AAV8, AAV-Spark100, AAV-LK03	60, 91 –94
Lung	AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV9	95 –98
Retina	AAV2, AAV4, AAV5, AAV8	63, 99
Skeletal muscle	AAV1, AAV6, AAV8, AAV9	55 –57, 100

AAV, adeno-associated virus.

AAV-Mediated Non-Homologous Integration and HR

Previous studies have demonstrated that wild-type AAV can integrate into a site at chromosome 19q13.4, termed the AAVS1 locus, in the absence of a helper virus.¹⁰ More than 70% of integration events preferentially occur at this site via DSB and non-homologous end joining (NHEJ) pathways, which can induce insertions, deletions, mutations, or chromosomal translocations. Rep78/Rep68 play a major role in DSB, which can mediate the complex formation between AAV ITRs and the chromosome 19 integration site.¹¹ AAVS1 is considered a safe harbor for the integration of a foreign gene. Therefore, antibiotic resistance genes (e.g., neo) or fluorescent genes (e.g., eGFP) can be integrated into the AAVS1 locus for further studies, which can provide convenient markers for the specific selection of transduced cells.¹² AAV-mediated non-homologous integration enables the precise engineering of new cell lines. Moreover, rAAVs do not cause genomic breaks themselves, which is a benefit for their clinical use.¹³ But rAAVs can still integrate at existing DSBs sites at the same time.¹³ However, to reach more precise gene targeting or gene therapy, HR may be the better choice rather than AAV-mediated non-homologous integration.

HR is an accurate genome modification method, although it generally occurs at a low efficiency of 10^–8–10^–6. Compared to traditional methods based on transfection or electroporation of plasmid constructs, AAV-mediated site-specific integration frequency is 1,000-fold higher, reaching approximately 1% at the single-copy hypoxanthine phosphoribosyl transferase (HPRT) locus in normal human fibroblasts.¹⁴ The HR frequency also varies in mammalian cells, depending on the genetic locus.¹⁵ Additionally, HR frequency is sensitive to single-nucleotide polymorphisms (SNPs), and even a single mismatch can alter HR frequency. Hence, SNPs can be harnessed for directing allele-specific targeting in certain situations.¹⁶

By altering the DNA sequence between homology arms, site-specific HR can be achieved. AAV-mediated HR has been shown to be able to introduce DNA substitutions, insertions, and deletion mutations with high fidelity at multiple chromosomal positions.^14,17 However, there is a preference for HR to occur at active transcriptional units because of the exposed single DNA strand.¹⁸ To deliver an additional gene to correct mutations or to produce disease models for basic research or preclinical gene therapy, AAV methodology is of great value. Hirata et al. used AAV to introduce a 1.5 kb transgene cassette into HPRT and COL1A1 (type I collagen) loci in normal human fibroblasts with an efficiency of 1%.¹⁹ Miller et al. showed that AAV can accurately correct chromosomal mutations in vivo at frequencies of 10^–4–10^–5 in hepatocytes.²⁰ Precise gene correction was observed by injecting AAV vectors carrying a lacZ fragment using the ROSA26 knock-in mouse with a missense mutation in lacZ.²⁰ Furthermore, a Duchenne muscular dystrophy (DMD) mouse model (mdx) was created by HR following intravascular co-delivery with two mini dystrophin-encoding rAAV6 vectors.²¹ In order to reach the standard required for clinical therapeutic applications, the efficiency of HR is a crucial problem that must be overcome. As a result, new rAAV-rDNA vectors were created whose expression cassette is flanked by 28S ribosomal DNA (rDNA) sequences capable of HR into genomic rDNA.^22,23 Surprisingly, incorporation of a ribosome-skipping 2A peptide coding sequence into AAV-based targeting vectors for promoter trapping can dramatically improve gene-targeting efficiencies.²⁴ Blocking of undesired repair pathways such as NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK) or Ku70 can also elevate HR frequency more than AAV alone.^25,26 Recently, a new AAV serotype, AAVHSC, is of remarkably higher infective and recombinant efficiency in CD34+ stem cells and some post-mitotic cells, which can enable the therapeutic manipulation of gene-based diseases.^27,28

Based on the above results, some pioneering studies addressing genetic diseases have been performed. Chamberlain et al. used AAV contained an IRES-neo cassette to disrupt dominant mutant COL1A1 collagen genes in mesenchymal stem cells from osteogenesis imperfecta sufferings.²⁹ Mucopolysaccharidosis type VII or Sly syndrome is an inherited disease caused by the mutation of GusB gene, and this was corrected at a frequency of up to 10^–4 through AAV-mediated HR.²⁹ Also, a hereditary tyrosinemia type I mouse model (Fah^5981SB ) was used to evaluate gene repair by delivering an AAV containing the Fah sequence (AAV-Fah^5981SB ), and a correction frequency of 10^–3 was achieved.³⁰ Epidermolysis bullosa (EB) is an inherited skin disease that is linked to dominantly inherited mutations in KRT5 or KRT14. Petek et al. used an AAV-mediated gene-targeting approach to correct KRT14 mutations in epidermal stem cells.³¹ In addition, Li et al. introduced a TKNEO cassette into the APP gene in trisomy 21 induced pluripotent stem cells through AAV-mediated HR and removed the extra copy of chromosome 21.³² Moreover, an AAV containing the gene for human coagulation factor IX (hF9) was used to infect mice with type B hemophilia, achieving on-target integration into ∼0.5% of the albumin alleles in hepatocytes and displaying therapeutic effects.³³ The outcomes described in these studies undoubtedly push AAV-mediated HR clearly into the clinical research arena, despite the ongoing challenges of HR efficiency.

AAV-Mediated Genome editing with Nucleases

To enhance the frequency of AAV-mediated HR, nucleases that can trigger DSB can be combined with the AAV to enhance HR activity. Previous studies confirmed that DSB played an important stimulatory role in genome editing and could increase AAV-mediated gene-targeting frequencies drastically by many thousand-fold. Thus, AAV-mediated genome editing with additional nucleases has developed into an efficient genetic tool, which enables the generation disease models and gene therapy agents.

So far, site-specific nucleases, including ZFN, TALEN, and CRISPR-Cas9, have led to breakthroughs in genome editing. CRISPR-Cas9 is the most popular of these because of its simplicity, efficiency, and low cost. Site-specific genome editing allows target genes to be knocked in, knocked out, or knocked down, and mutations can be restored effectively through AAV-mediated delivery.

Previous studies have demonstrated that systemic co-delivery of ZFN (or AAV-ZFN, ZFN mRNA) and AAV donor template works effectively in many situations. For reporter system construction, ZFN mRNA and AAV6 donor template were delivered into human hematopoietic stem cells (HSCs) and progenitor cells, resulting in the site-specific insertion of a promoter-GFP cassette.³⁴ In the preclinical gene therapy setting, ZFN and a G6PC donor transgene, both delivered with AAV, were targeted to the ROSA26 safe-harbor locus, markedly improving the survival of G6Pase knockout (G6Pase-KO) mice.³⁵ In a mouse model of hemophilia B, injection of an AAV encoding ZFN (AAV-ZFN) targeting the human F9 gene and AAV donor template (AAV-hF9) led to in situ correction of a defective hF9 gene and measurable circulating factor IX levels.^36,37 Moreover, AAV-mediated delivery of ZFNs individually targeted to hepatitis B virus (HBV) genes has an impact on HBV DNA replication and reduces the production of infectious HBV virions, extending this approach to new strategies to resolve infectious diseases.³⁸ Hitherto, ZFNs packed within AAV2/6 were applied in clinical trials for hemophilia B (NCT02695160), mucopolysaccharidosis I (NCT02702115), and mucopolysaccharidosis II (NCT03041324; ClinicalTrials.gov). AAV-mediated genome editing with ZFN has the potential to evolve into a sophisticated pharmaceutical to treat hereditary diseases.

Although ZFN technology is the only genome-editing tool applied in the clinical gene therapy arena so far, off-target effects are measurable in preclinical studies, and this may be a concern.^39,40 TALEN, the second-generation genome-editing nuclease, is of lower toxicity than ZFN.⁴¹ Chapdelaine et al. developed an AAV9 coding for one TALE coupled with a transcription activation domain to increase gene expression.⁴² However, the large size (∼6.0 kb) and complex design of this construct render it comparatively difficult to package into one AAV, which restricts its application. In recent years, CRISPR-Cas9 has emerged as a more promising genome-editing tool with higher efficiency, and has been harnessed for genome editing in various species from microorganisms to plants and mammals. CRISPR-Cas9 has been applied in various fields, including disease model generation, dissection of disease mechanisms, and the development of preclinical or clinical therapies. Delivery to the appropriate target sites is essential for the practical application of this approach. However, traditional plasmid transfection is not effective in many cell types. In order to deliver a therapeutic gene editing construct to a wide variety cells in vivo or in vitro, AAV-based CRISPR-Cas9 delivery has been developed.⁴³ This takes advantage of the low immunogenicity of AAV, and since the CRISPR-Cas9 system is composed of two segments, Cas9 and sgRNA, delivery can be flexible according to different requirements.

Using Streptococcus pyogenes Cas9 (SpCas9), Swiech et al. designed a dual-vector system that packages SpCas9 (AAV-SpCas9) and sgRNA expression cassettes (AAV-SpGuide) in two separate viral vectors. They showed conspicuous disruption of multiple genes in post-mitotic neurons in adult mice.⁴⁴ Nishiyama et al. achieved highly efficient HR-based genome editing in developing and adult brains and in various brain areas of mouse in vivo through AAV-SpCas9 and AAV-donor-sgRNA.⁴⁵ Nevertheless, the size of the SpCas9 coding sequence (∼4.1 kb) limits its basic and clinical application as a result of the limited transgene capacity of AAV (∼4.7 kb).⁴⁶ Therefore, the subsequent discovery of the smaller-sized Staphylococcus aureus Cas 9 (SaCas9, ∼3.2 kb) has allowed its application in genome engineering. By packaging SaCas9 and its sgRNA expression cassette into a single AAV, a ∼95% decrease in serum proprotein convertase subtilisin/kexin type 9 (Pcsk9) and a ∼40% decrease in total cholesterol was observed 1 week after administration in mice.⁴⁷ Friedland et al. made use of two sgRNAs and SaCas9 packaging in a single AAV, which opened the door to multiple gene editing approaches using “all-in-one” AAV vectors.⁴⁸ In addition to AAV-CRISPR-Cas9-mediated knockout, knockdown and knock-in can also be realized by CRISPR-Cas9 and AAV donor. NRL, the gene for a key transcription factor in rod photoreceptor cells was knocked in by CRISPR-Cas9 and an AAV donor template in human embryonic stem cells (hESCs).⁴⁹ This established a platform for human genome manipulation in hESCs. Also, a large transgene cassette (∼6.5 kb) can be integrated into the genome by consecutive HR events using two separate AAV donors and CRISPR-Cas9.⁵⁰ To enhance the efficiency of CRISPR-Cas9-mediated gene disruption or HR, Gwiazda et al. utilized mRNA expressing Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with AAV vectors expressing guide RNAs and recombination templates. This was named an mRNA/AAV delivery system, and worked effectively in primary human T cells.⁵¹ Notably, homology-independent targeted integration (HITI) through AAV-SpCas9 and AAV-HITI (donor) is a new strategy for foreign gene knock-in. Using this approach, the retinal degeneration condition retinitis pigmentosa was treated in a rat model with a higher efficiency than traditional HR of 30–50%.⁵² Furthermore, an AAV-SpCas9 and AAV-donor-sgRNA strategy was devised to perform homology-mediated end joining (HMEJ)-based targeted gene integration, with an efficiency much greater than strategies based on HR, NHEJ, and microhomology-mediated end joining.⁵³ Both the HITI and HMEJ methods function efficiently in dividing and nondividing cells in vitro and in vivo.

With the favorable progress in AAV-mediated CRISPR-Cas9 genome editing, preclinical studies of various diseases have become increasingly common. In a DMD mouse model, a frameshift mutation in Dmd exon 23 was targeted through AAV-SpCas9 coupled with guide RNAs packed in one AAV, which led to the excision of exon 23 and restored the normal dystrophin reading frame.^54
–56 Therefore, by using a similar approach, Dmd exons 52 and 53 in dystrophic mdx^4cv mice were targeted, which made a new reading frame by deleting two exons and improved muscle function.⁵⁷ The smallest Cas9 ortholog, Campylobacter jejuni Cas9 (CjCas9, ∼3.0 kb) carrying sgRNA targeted to exon 23 was packed into a single AAV9 vector to treat a Dmd knockout mouse model with restoration of the reading frame through NHEJ.⁵⁸ Through two independent AAV9-SaCas9-sgRNAs targeting Lama2 in a mouse model of congenital muscular dystrophy type 1A (MDC1A), an intronic region containing the mutation was excised and a functional donor splice site was created through NHEJ.⁵⁹ Moreover, by utilizing the dual AAV system (AAV-Cas9, AAV-[donor]-sgRNA), an OTC gene mutation was corrected in newborn mice with hyperammonemia, a metabolic liver disease, at a frequency of approximately 10%⁶⁰; huntingtin (HTT) gene expression was reduced effectively in fibroblast cell lines derived from Huntington's disease patients⁶¹; an intronic fragment of the Cep290 gene related to LCA10 in the mouse retina was deleted⁶²; the Nrl gene in rods in three different mouse models of retinal degeneration was disrupted⁶³; the genomic VEGFR2 locus related to angiogenesis-associated diseases was edited to abrogate angiogenesis in mouse models of neovascularization⁶⁴; the mutant amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer's disease was targeted to decrease amyloid-β (Aβ) levels after the injection to hippocampus of adult mice⁶⁵; human-derived alpha-1 antitrypsin mutations (PiZ) were corrected by AAV-Cas9 and AAV-donor-sgRNA in a PiZ mouse model⁶⁶; GUCY2D mutations of LCA were disrupted in mouse and macaque photoreceptor without Cas9-specific T-cell responses, demonstrating the viability of treating inherited retinal diseases.⁶⁷ The achievements outlined above manifested appreciable therapeutic benefits relevant to human genetic disease, and reveal that the dual AAV system (AAV-Cas9, AAV-[donor]-sgRNA) is a practicably applicable method to mediate genome editing. Due to the off-target effects of CRISPR-Cas9, Cas9 ribonucleoprotein (RNP) and rAAV6 (donor) were combined to achieve HR at the HBB gene in HSCs at a frequency of 22.5%, with lower off-target effects.⁶⁸ AAV-mediated genome editing with CRISPR-Cas9 can also be applied to non-inherited diseases as well as inherited diseases using an “all-in-one” AAV. For instance, CjCas9 targeted Vegfa was to treat AMD in the mouse retina by injecting AAV-CjCas9-sgRNA in one AAV.⁶⁹ By using SaCas9 and multiple sgRNAs in one AAV, the HIV-1 provirus was excised in three different animal models, providing a potential approach for AIDS therapy.⁷⁰ To inhibit HBV replication, AAV8-SaCas9-sgRNA was used to decrease the levels of serum HBsAg and HBeAg in HBV transgenic mice.⁷¹ Notably, the small effective capacity of AAV is a huge obstacle that limits the application of some genome-editing tools (Table 3).

Table 3.

Naturally occurring genome-editing nucleases

Nucleases	ORF (kb)	PAM	References
AsCpf1	3.9	TTTN	101
CjCas9	3.0	NNNNRYAC	69
FnCas9	4.9	NGG	102
FnCpf1	3.9	KYTV	103
LbCfp1	3.7	TTTN	101
NmCas9	3.2	NNNNGATT	104
SaCas9	3.2	NNGRRT	47
SpCas9	4.1	NGG	105
St1Cas9	3.4	NNAGAAW	106
St3Cas9	4.2	NGGNG	107

ORF, open reading frame; PAM, protospacer adjacent motif.

AAV-mediated genome editing with nucleases, especially CRISPR-Cas9, has led to rapid developments in the gene therapy field. However, how efficiency could be enhanced up to the standards needed for effective clinical treatment in terms of both AAV delivery and nuclease activity should be addressed. Furthermore, the off-target effects induced by nucleases are challenging. With the identification of high-fidelity Cas9 variants and base-editing enzymes such as cytidine deaminases or adenine deaminase, off-target effects may be minimized.^72,73

Conclusions and Outlooks

AAV is considered a promising gene delivery vector for genome editing and is widely used in a variety of studies. Compared to other viral and non-viral vectors, AAV is safe and efficient for human gene therapy applications. There are two major approaches to AAV-mediated genome editing: HR and combination with nucleases. HR is accurate, while its efficiency is pretty low. However, by combining the two methods, further efficient genome editing can be carried out with the donor template and nucleases to repair mutations. For certain diseases caused by overexpressed genes, only nucleases would be needed for disruption. Moreover, with the engineering of nucleases, base editing (C to T or A to G) has now been realized, and these types of alterations contribute a lot to single-base mutation diseases.^72,73 AAV-mediated base editing could be realized and single-base mutations could be corrected with the modulation of its package size.

Owing to the variety of AAV serotypes, AAV vectors have been used to infect various tissues successfully, including the retina, liver, heart, skeletal muscle, central nervous system, and so on through different modes of injection. As a result, rAAV is commonly used in gene therapy. Although many preclinical studies and clinical trials of AAV-mediated gene therapy have been performed, problems and obstacles (capacity, efficiency, presence of neutralizing antibodies, and off-target effects) still exist. Notably, the risk of hepatocellular carcinoma of AAV2 is reported, possibly due to the presence of a liver-specific enhancer-promoter element in the wild-type AAV2 genome,⁷⁴ which raises more concerns for the selection of the promoter in the constructs.

The capacity of AAV vector is up to 4.7 kb with workable efficiency,⁴⁶ which limits the application of some genome-editing tools. In order to enlarge the capacity of AAV vector, a set of efficient trans-splicing vectors for the transfection of large therapeutic genes were generated.⁷⁵ An intein-split system for the generation of the full-length protein after protein trans-splicing was applied into AAV base editors (AAV8.N-int-BE3 and AAV8.C-int-BE3.sgRNA), which was utilized for the treatment of a metabolic disease in adult mice.^76,77 However, in spite of the dual AAV system and use of RNP, efficiency may not be sufficient to apply to clinical gene therapy. Infection efficiency is also a crucial problem. Therefore, new methods have been developed for the robust infection to the target tissues. For this purpose, novel AAV capsids have been created to alter tissue tropism. For example, an AAV2/8 chimera was engineered, which can transfer genes to cardiac and skeletal muscle tissues with high efficiency and has decreased hepatic tropism.⁷⁸ An engineered AAV2/9 chimera displays preferential and robust neuronal transduction within the brain and decreased glial tropism.⁷⁹ Anc80L65, a synthetic AAV vector, enabled highly efficient transduction of ocular inner and outer hair cells, benefiting gene therapy approaches for hearing and balance disorders.⁸⁰ Recently, two novel AAV vectors (AAV-PHP.eB and AAV-PHP.S) were designed that could efficiently transduce to the central and peripheral nervous systems, respectively.⁸¹ It is encouraging that different tissue-specific AAV serotypes were exploited, which has raised hopes for the treatment of different diseases.

The optimization of AAV vectors described above enables potential and extensive applications in gene therapy (Table 4). More importantly, with the combination of nucleases, AAV-mediated genome editing has shown increasing promise in basic and clinical applications. Unlike AAV carrying therapeutic genes present as episomes, AAV-mediated genome editing with nucleases can realize in situ genetic correction, even at the resolution of single base correction. Although the presence of neutralizing antibodies to AAV vectors and nucleases is an existing problem that is yet to be solved⁸² and elimination of off-target effects is also needed as much as possible, AAV vectors and nucleases can be fully exploited for in vivo and in situ gene correction for the clinical treatment of human diseases.

Table 4.

Problems and possible strategies of AAV-mediated genome editing

Problems	Possible strategies
Low capacity	1. Dual AAV system (nuclease and [donor]-sgRNA)
	2. Detection and optimization for the smallest nucleases
	3. Engineering of AAV vectors
	4. Intein-split method
Efficiency	1. rAAV-rDNA vectors
	2. Incorporation of a ribosome-skipping 2A peptide coding sequence
	3. Inhibition of undesired pathways (NHEJ)
	4. Tissue specific serotypes
	5. Combination with the nucleases
Off-targets (nuclease)	1. High-fidelity Cas9 variants
	2. Base-editing nucleases
	3. RNP and rAAV-(donor)-sgRNA method
Neutralizing antibodies	1. Exploration of novel AAV serotypes
Neutralizing antibodies	2. Targeted gene therapy

sgRNA, single guide RNA; rAAV, recombinant AAV; NHEJ, non-homologous end joining; RNP, ribonucleoprotein.

Footnotes

Acknowledgments

This work was supported by grants from the Natural Science Foundation of China (81201181; F.G.), Science Technology project of Zhejiang Province (2017C37176; F.G.).

Author Disclosure

All authors declare that they have no competing interests.

References

Ballinger

, Goode

, Ray-Coquard

, et al. Monogenic and polygenic determinants of sarcoma risk: an international genetic study. Lancet Oncol, 2016; 17:1261–1271.

Ferreira

, Twisk

, Kwikkers

, et al. Immune responses to intramuscular administration of alipogene tiparvovec (AAV1-LPL(S447X)) in a Phase II clinical trial of lipoprotein lipase deficiency gene therapy. Hum Gene Ther, 2014; 25:180–188.

Nathwani

, Reiss

, Tuddenham

, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med, 2014; 371:1994–2004.

MacLaren

, Groppe

, Barnard

, et al. Retinal gene therapy in patients with choroideremia: initial findings from a Phase 1/2 clinical trial. Lancet, 2014; 383:1129–1137.

Pleger

, Shan

, Ksienzyk

, et al. Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model. Sci Transl Med, 2011; 3:92ra64.

Heier

, Kherani

, Desai

, et al. Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: a Phase 1, open-label trial. Lancet, 2017; 390:50–61.

Hirsch

, Samulski

. AAV-mediated gene editing via double-strand break repair. Methods Mol Biol, 2014; 1114:291–307.

Xie

, Bu

, Bhatia

, et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci U S A, 2002; 99:10405–10410.

Sonntag

, Schmidt

, Kleinschmidt

. A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc Natl Acad Sci U S A, 2010; 107:10220–10225.

10.

Henckaerts

, Dutheil

, Zeltner

, et al. Site-specific integration of adeno-associated virus involves partial duplication of the target locus. Proc Natl Acad Sci U S A, 2009; 106:7571–7576.

11.

Lamartina

, Ciliberto

, Toniatti

. Selective cleavage of AAVS1 substrates by the adeno-associated virus type 2 rep68 protein is dependent on topological and sequence constraints. J Virol, 2000; 74:8831–8842.

12.

Smith

, Maguire

, Davis

, et al. Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem cells, 2008; 26:496–504.

13.

Miller

, Petek

, Russell

. Adeno-associated virus vectors integrate at chromosome breakage sites. Nat Genet, 2004; 36:767–773.

14.

Russell

, Hirata

. Human gene targeting by viral vectors. Nat Genet, 1998; 18:325–330.

15.

Yanez

, Porter

. A chromosomal position effect on gene targeting in human cells. Nucleic Acids Res, 2002; 30:4892–4901.

16.

Deyle

, Li

, Ren

, et al. The effects of polymorphisms on human gene targeting. Nucleic Acids Res, 2014; 42:3119–3124.

17.

Inoue

, Dong

, Hirata

, et al. Introduction of single base substitutions at homologous chromosomal sequences by adeno-associated virus vectors. Mol Ther, 2001; 3:526–530.

18.

Deyle

, Hansen

, Cornea

, et al. A genome-wide map of adeno-associated virus-mediated human gene targeting. Nat Struct Mol Biol, 2014; 21:969–975.

19.

Hirata

, Chamberlain

, Dong

, et al. Targeted transgene insertion into human chromosomes by adeno-associated virus vectors. Nat Biotechnol, 2002; 20:735–738.

20.

Miller

, Wang

, Petek

, et al. Gene targeting in vivo by adeno-associated virus vectors. Nat Biotechnol, 2006; 24:1022–1026.

21.

Odom

, Gregorevic

, Allen

, et al. Gene therapy of mdx mice with large truncated dystrophins generated by recombination using rAAV6. Mol Ther, 2011; 19:36–45.

22.

Lisowski

, Lau

, Wang

, et al. Ribosomal DNA integrating rAAV-rDNA vectors allow for stable transgene expression. Mol Ther, 2012; 20:1912–1923.

23.

Wang

, Lisowski

, Finegold

, et al. AAV vectors containing rDNA homology display increased chromosomal integration and transgene persistence. Mol Ther, 2012; 20:1902–1911.

24.

Karnan

, Ota

, Konishi

, et al. Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide. Nucleic Acids Res, 2016; 44:e54.

25.

Fattah

, Lichter

, Fattah

, et al. Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells. Proc Natl Acad Sci U S A, 2008; 105:8703–8708.

26.

Paulk

, Loza

, Finegold

, et al. AAV-mediated gene targeting is significantly enhanced by transient inhibition of nonhomologous end joining or the proteasome in vivo . Hum Gene Ther, 2012; 23:658–665.

27.

Smith

, Ul-Hasan

, Carvaines

, et al. Gene transfer properties and structural modeling of human stem cell-derived AAV. Mol Ther, 2014; 22:1625–1634.

28.

Smith

, Wright

, Clark

, et al. Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing. Proc Natl Acad Sci U S A, 2018; 115:E7379–E7388.

29.

Chamberlain

, Schwarze

, Wang

, et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science, 2004; 303:1198–1201.

30.

Paulk

, Wursthorn

, Wang

, et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo . Hepatology, 2010; 51:1200–1208.

31.

Petek

, Fleckman

, Miller

. Efficient KRT14 targeting and functional characterization of transplanted human keratinocytes for the treatment of epidermolysis bullosa simplex. Mol Ther, 2010; 18:1624–1632.

32.

, Chang

, Wang

, et al. Trisomy correction in Down syndrome induced pluripotent stem cells. Cell Stem Cell, 2012; 11:615–619.

33.

Barzel

, Paulk

, Shi

, et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature, 2015; 517:360–364.

34.

Wang

, Exline

, DeClercq

, et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol, 2015; 33:1256–1263.

35.

Landau

, Brooks

, Perez-Pinera

, et al. In vivo zinc finger nuclease-mediated targeted integration of a glucose-6-phosphatase transgene promotes survival in mice with glycogen storage disease type IA. Mol Ther, 2016; 24:697–706.

36.

Anguela

, Sharma

, Doyon

, et al. Robust ZFN-mediated genome editing in adult hemophilic mice. Blood, 2013; 122:3283–3287.

37.

, Haurigot

, Doyon

, et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature, 2011; 475:217–221.

38.

Weber

, Stone

, Sedlak

, et al. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication. PLoS One, 2014; 9:e97579.

39.

Gupta

, Meng

, Zhu

, et al. Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases. Nucleic Acids Res, 2011; 39:381–392.

40.

Sander

, Ramirez

, Linder

, et al. In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites. Nucleic Acids Res, 2013; 41:e181.

41.

Mussolino

, Alzubi

, Fine

, et al. TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res, 2014; 42:6762–6773.

42.

Chapdelaine

, Gerard

, Sanchez

, et al. Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice. Gene Ther, 2016; 23:606–614.

43.

Senis

, Fatouros

, Grosse

, et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol J, 2014; 9:1402–1412.

44.

Swiech

, Heidenreich

, Banerjee

, et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol, 2015; 33:102–106.

45.

Nishiyama

, Mikuni

, Yasuda

. Virus-mediated genome editing via homology-directed repair in mitotic and postmitotic cells in mammalian brain. Neuron, 2017; 96:755–768.e5.

46.

, Yang

, Colosi

. Effect of genome size on AAV vector packaging. Mol Ther, 2010; 18:80–86.

47.

Ran

, Cong

, Yan

, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature, 2015; 520:186–191.

48.

Friedland

, Baral

, Singhal

, et al. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol, 2015; 16:257.

49.

, Xi

, Yang

, et al. CRISPR/Cas9-AAV mediated knock-in at NRL locus in human embryonic stem cells. Mol Ther Nucleic Acids, 2016; 5:e393.

50.

Bak

, Porteus

. CRISPR-mediated integration of large gene cassettes using AAV donor vectors. Cell Rep, 2017; 20:750–756.

51.

Gwiazda

, Grier

, Sahni

, et al. High efficiency CRISPR/Cas9-mediated gene editing in primary human T-cells using mutant adenoviral E4orf6/E1b55k “helper” proteins. Mol Ther, 2016; 24:1570–1580.

52.

Suzuki

, Tsunekawa

, Hernandez-Benitez

, et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature, 2016; 540:144–149.

53.

Yao

, Wang

, Hu

, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res, 2017; 27:801–814.

54.

Long

, Amoasii

, Mireault

, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science, 2016; 351:400–403.

55.

Nelson

, Hakim

, Ousterout DG

, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science, 2016; 351:403–407.

56.

Tabebordbar

, Zhu

, Cheng

JKW

, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science, 2016; 351:407–411.

57.

Bengtsson

, Hall

, Odom

, et al. Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nat Commun, 2017; 8:14454.

58.

Koo

, Lu-Nguyen

, Malerba

, et al. Functional rescue of dystrophin deficiency in mice caused by frameshift mutations using Campylobacter jejuni Cas9. Mol Ther, 2018; 26:1529–1538.

59.

Kemaladewi

, Maino

, Hyatt

, et al. Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism. Nat Med, 2017; 23:984–989.

60.

Yang

, Wang

, Bell

, et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol, 2016; 34:334–338.

61.

Monteys

, Ebanks

, Keiser

, et al. CRISPR/Cas9 editing of the mutant huntingtin allele in vitro and in vivo . Mol Ther, 2017; 25:12–23.

62.

Ruan

, Barry

, Yu

, et al. CRISPR/Cas9-mediated genome editing as a therapeutic approach for Leber congenital amaurosis 10. Mol Ther, 2017; 25:331–341.

63.

, Mookherjee

, Chaitankar

, et al. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun, 2017; 8:14716.

64.

Huang

, Zhou

, Wu

, et al. Genome editing abrogates angiogenesis in vivo . Nat Commun, 2017; 8:112.

65.

Gyorgy

, Loov

, Zaborowski

, et al. CRISPR/Cas9 mediated disruption of the Swedish APP allele as a therapeutic approach for early-onset Alzheimer's disease. Mol Ther Nucleic Acids, 2018; 11:429–440.

66.

Song

, Wang

, Jiang

, et al. In vivo genome editing partially restores alpha1-antitrypsin in a murine model of AAT deficiency. Hum Gene Ther, 2018; 29:853–860.

67.

McCullough

, Boye

, Fajardo

, et al. Somatic gene editing of GUCY2D by AAV-CRISPR/Cas9 alters retinal structure and function in mouse and macaque. Hum Gene Ther, 2018 Oct 25 [Epub ahead of print]; DOI: 10.1089/hum.2018.193.

68.

Dever

, Bak

, Reinisch

, et al. CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature, 2016; 539:384–389.

69.

Kim

, Koo

, Park

, et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni . Nat Commun, 2017; 8:14500.

70.

Yin

, Zhang

, Qu

, et al. In vivo excision of HIV-1 provirus by saCas9 and multiplex single-guide RNAs in animal models. Mol Ther, 2017; 25:1168–1186.

71.

, Sheng

, Liu

, et al. Inhibition of HBV expression in HBV transgenic mice using AAV-delivered CRISPR-SaCas9. Front Immunol, 2018; 9:2080.

72.

Gaudelli

, Komor

, Rees

, et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature, 2017; 551:464–471.

73.

Kim

, Komor

, Levy

, et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol, 2017; 35:371–376.

74.

Logan

, Dane

, Hallwirth

, et al. Identification of liver-specific enhancer-promoter activity in the 3′ untranslated region of the wild-type AAV2 genome. Nat Genet, 2017; 49:1267–1273.

75.

Lai

, Yue

, Liu

, et al. Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors. Nat Biotechnol, 2005; 23:1435–1439.

76.

Truong

, Kühner

, Kühn

, et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res, 2015; 43:6450–6458.

77.

Villiger

, Grisch-Chan

, Lindsay

, et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat Med, 2018; 24:1519–1525.

78.

Asokan

, Conway

, Phillips

, et al. Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle. Nat Biotechnol, 2010; 28:79–82.

79.

Murlidharan

, Sakamoto

, Rao

, et al. CNS-restricted transduction and CRISPR/Cas9-mediated gene deletion with an engineered AAV vector. Mol Ther Nucleic Acids, 2016; 5:e338.

80.

Landegger

, Pan

, Askew

, et al. A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear. Nat Biotechnol, 2017; 35:280–284.

81.

Chan

, Jang

, Yoo

, et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci, 2017; 20:1172–1179.

82.

Moreno

, Palmer

, Aleman

, et al. Exploring protein orthogonality in immune space: a case study with AAV and Cas9 orthologs. bioRxiv, 2018 Jan 10 [Epub ahead of print]; DOI: 10.1101/245985.

83.

Di Meo

, Marchet

, Lamperti

, et al. AAV9-based gene therapy partially ameliorates the clinical phenotype of a mouse model of Leigh syndrome. Gene Ther, 2017; 24:661–667.

84.

Laoharawee

, Podetz-Pedersen

, Nguyen

, et al. Prevention of neurocognitive deficiency in mucopolysaccharidosis type II mice by central nervous system-directed, AAV9-mediated iduronate sulfatase gene transfer. Hum Gene Ther, 2017; 28:626–638.

85.

Morabito

, Giannelli

, Ordazzo

, et al. AAV-PHP.B-mediated global-scale expression in the mouse nervous system enables GBA1 gene therapy for wide protection from synucleinopathy. Mol Ther, 2017; 25:2727–2742.

86.

Rincon

, de Vin

, Duque

, et al. Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector. Gene Ther, 2018; 25:83–92.

87.

Balazs

, Chen

, Hong

, et al. Antibody-based protection against HIV infection by vectored immunoprophylaxis. Nature, 2011; 481:81–84.

88.

Gardner

, Kattenhorn

, Kondur

, et al. AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature, 2015; 519:87–91.

89.

Ruozi

, Bortolotti

, Falcione

, et al. AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia. Nat Commun. 2015; 6:7388.

90.

Wang

, Zhu

, Qiao

, et al. Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol, 2005; 23:321–328.

91.

George

, Sullivan

, Giermasz

, et al. Hemophilia B gene therapy with a high-specific-activity factor IX variant. N Engl J Med, 2017; 377:2215–2227.

92.

Lisowski

, Dane

, Chu

, et al. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature, 2014; 506:382–386.

93.

Nault

, Datta

, Imbeaud

, et al. Adeno-associated virus type 2 as an oncogenic virus in human hepatocellular carcinoma. Mol Cell Oncol, 2016; 3:e1095271.

94.

Rezvani

, Espanol-Suner

, Malato

, et al. In vivo hepatic reprogramming of myofibroblasts with AAV vectors as a therapeutic strategy for liver fibrosis. Cell Stem Cell, 2016; 18:809–816.

95.

Chen

, Akerstrom

, Baus

, et al. Comparative analysis of the transduction efficiency of five adeno associated virus serotypes and VSV-G pseudotype lentiviral vector in lung cancer cells. J Virol, 2013; 10:86.

96.

Hayes

, Curley

, Masterson

, et al. Pulmonary overexpression of inhibitor kappaBalpha decreases the severity of ventilator-induced lung injury in a rat model. Br J Anaesth, 2014; 113:1046–1054.

97.

Limberis

, Vandenberghe

, Zhang

, et al. Transduction efficiencies of novel AAV vectors in mouse airway epithelium in vivo and human ciliated airway epithelium in vitro . Mol Ther, 2009; 17:294–301.

98.

Limberis

, Wilson

. Adeno-associated virus serotype 9 vectors transduce murine alveolar and nasal epithelia and can be readministered. Proc Natl Acad Sci U S A, 2006; 103:12993–12998.

99.

Wiley

, Burnight

, Kaalberg

, et al. Assessment of adeno-associated virus serotype tropism in human retinal explants. Hum Gene Ther, 2018; 29:424–436.

100.

Mendell

, Sahenk

, Malik

, et al. A Phase 1/2a follistatin gene therapy trial for Becker muscular dystrophy. Mol Ther, 2015; 23:192–201.

101.

Zetsche

, Gootenberg

, Abudayyeh

, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015; 163:759–771.

102.

Sampson

, Saroj

, Llewellyn

, et al. A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature, 2013; 497:254–257.

103.

, Lin

, Cheng

, et al. A “new lease of life”: FnCpf1 possesses DNA cleavage activity for genome editing in human cells. Nucleic Acids Res, 2017; 45:11295–11304.

104.

Esvelt

, Mali

, Braff

, et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods, 2013; 10:1116–1121.

105.

Cong

, Ran

, Cox

, et al. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013; 339:819–823.

106.

Garneau

, Dupuis

, Villion

, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010; 468:67–71.

107.

Gasiunas

, Barrangou

, Horvath

, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A, 2012; 109:E2579–2586.

Basic and Clinical Application of Adeno-Associated Virus–Mediated Genome Editing

Abstract

Introduction

AAV Structure and Serotypes

AAV-Mediated Non-Homologous Integration and HR

AAV-Mediated Genome editing with Nucleases

Conclusions and Outlooks

Footnotes

Acknowledgments

Author Disclosure

References