Comparison of dystrophin expression following gene editing and gene replacement in an aged preclinical DMD animal model.

Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young animals with limited muscle fibrosis and wasting, thereby favoring muscle transduction, myonuclear editing, and prevention of disease progression. Here, we explore muscle-specific dystrophin gene editing following intramuscular delivery of AAV6:CK8e-CRISPR/SaCas9 in 3- and 8-year-old dystrophic CXMD dogs and provide a qualitative comparison to AAV6:CK8e-micro-dystrophin gene replacement at 6 weeks post-treatment. Gene editing restored the dystrophin reading frame in ∼1.3% of genomes and in up to 4.0% of dystrophin transcripts following excision of a 105-kb mutation containing region spanning exons 6-8. However, resulting dystrophin expression levels and effects on muscle pathology were greater with the use of micro-dystrophin gene transfer. This study demonstrates that our muscle-specific multi-exon deletion strategy can correct a frequently mutated region of the dystrophin gene in an aged large animal DMD model, but underscores that further enhancements are required to reach efficiencies comparable to AAV micro-dystrophin. Our observations also indicate that treatment efficacy and state of muscle pathology at the time of intervention are linked, suggesting the need for additional methodological optimizations related to age and disease progression to achieve relevant clinical translation of CRISPR-based therapies to all DMD patients.

AAV6 Vector Production and Purification for Muscle Gene Therapy.

Vectors derived from adeno-associated viruses (AAV) have been generated using numerous naturally occurring and synthetic serotypes of the virus. Such vectors have proven to be extremely useful for a variety of gene transfer studies, both in vitro and in vivo, and are increasingly being used in gene therapy protocols for a variety of human disorders. Methods to produce AAV vectors typically rely on co-transfection of several different plasmid vectors that carry the transgene of interest (the gene to be delivered , in a "transfer plasmid") and helper genes needed for AAV vector replication and packaging (helper plasmids). While the methods used to generate AAV are conceptually simple, minor variations in a variety of steps can result in significant differences in the overall yield of vector. Here we describe protocols for generating vectors derived from AAV6, which are particularly useful for gene transfer to muscle tissues.

Successful regional delivery and long-term expression of a dystrophin gene in canine muscular dystrophy: a preclinical model for human therapies.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.

Lung cancer in mice induced by the jaagsiekte sheep retrovirus envelope protein is not maintained by rare cancer stem cells, but tumorigenicity does correlate with Wnt pathway activation.

JSRV, a simple beta-retrovirus, is the etiologic agent of ovine pulmonary adenocarcinoma, a form of non-small cell lung cancer in sheep and goats. It has been shown that the envelope protein alone is sufficient to induce tumorigenesis in the lungs of mice when delivered via an adeno-associated viral vector. Here, we tested the hypothesis that JSRV envelope-induced tumors are maintained by a small population of tumor-initiating cells, termed cancer stem cells. To test this hypothesis, dissociated cancer cells were sorted from envelope-induced tumors in mouse lung based on the putative stem cell markers Sca-1, CD34, and CD133, the pluripotency-associated transcription factor Oct4, and the level of Wnt signaling. No association with increased tumor-initiating capacity was found with any of the cell-surface markers. In addition, we were unable to detect any evidence of Oct4 expression in tumor-bearing mouse lung. However, tumor cells possessing an active Wnt signaling pathway did show a significant correlation with increased tumor formation upon transplantation. Limiting dilution transplant analysis suggests the existence of a large fraction of cells with the ability to propagate tumor growth, with increasing tumor initiation potential correlating with activated Wnt signaling.

Jaagsiekte sheep retrovirus and enzootic nasal tumor virus promoters drive gene expression in all airway epithelial cells of mice but only induce tumors in the alveolar region of the lungs.

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats, while the closely related enzootic nasal tumor virus type 1 (ENTV-1) and ENTV-2 induce tumors in the nasal epithelium of sheep and goats, respectively. When expressed using a strong Rous sarcoma virus promoter, the envelope proteins of these viruses induce tumors in the respiratory tract of mice, but only in the distal airway. To examine the role of the retroviral long terminal repeat (LTR) promoters in determining tissue tropism, adeno-associated virus (AAV) vectors expressing alkaline phosphatase under the control of the JSRV, ENTV-1, or ENTV-2 LTRs were generated and administered to mice. The JSRV LTR was active in all airway epithelial cells, while the ENTV LTRs were active in the nasal epithelium and alveolar type II cells but poorly active in tracheal and bronchial epithelial cells. When vectors were administered systemically, the ENTV-1 and -2 LTRs were inactive in major organs examined, whereas the JSRV showed high-level activity in the liver. When a putative transcriptional enhancer from the 3' end of the env gene was inserted upstream of the JSRV and ENTV-1 LTRs in the AAV vectors, a dramatic increase in transgene expression was observed. However, intranasal administration of AAV vectors containing any combination of ENTV or JSRV LTRs and Env proteins induced tumors only in the lower airway. Our results indicate that mice do not provide an adequate model for nasal tumor induction by ENTV despite our ability to express genes in the nasal epithelium.

Expression of human alpha1-antitrypsin in mice and dogs following AAV6 vector-mediated gene transfer to the lungs.

We evaluated the potential of lung-directed gene therapy for alpha1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans.

High-efficiency promoter-dependent transduction by adeno-associated virus type 6 vectors in mouse lung.

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues has been shown to depend heavily on the AAV type from which the vector capsid proteins are derived. Among the AAV types studied, AAV6 efficiently transduces cells of the airway epithelium, making it a good candidate for the treatment of lung diseases such as cystic fibrosis. Here we have evaluated the effects of various promoter sequences on transduction rates and gene expression levels in the lung. Of the strong viral promoters examined, the Rous sarcoma virus (RSV) promoter performed significantly better than a human cytomegalovirus (CMV) promoter in the airway epithelium. However, a hybrid promoter consisting of a CMV enhancer, beta-actin promoter and splice donor, and a beta-globin splice acceptor (CAG promoter) exhibited even higher expression than either of the strong viral promoters alone, showing a 38-fold increase in protein expression over the RSV promoter. In addition, we show that vectors containing either the RSV or CAG promoter expressed well in the nasal and tracheal epithelium. Transduction rates in the 90% range were achieved in many airways with the CAG promoter, showing that with the proper AAV capsid proteins and promoter sequences, efficient transduction can be achieved.

Envelope proteins of Jaagsiekte sheep retrovirus and enzootic nasal tumor virus induce similar bronchioalveolar tumors in lungs of mice.

Jaagsiekte sheep retrovirus (JSRV) induces bronchioalveolar tumors in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse airway epithelial cells induces similar tumors, indicating that Env expression is sufficient for tissue-specific tumor formation. Enzootic nasal tumor virus (ENTV) is related to JSRV but induces tumors in the nasal epithelium of sheep and goats. Here we found that ENTV Env can also induce tumors in mice but, unexpectedly, with a phenotype identical to that of tumors induced by the JSRV Env, indicating that factors other than Env mediate the tissue specificity of tumor induction by ENTV.

Prevalence of neutralizing antibodies against adeno-associated virus (AAV) types 2, 5, and 6 in cystic fibrosis and normal populations: Implications for gene therapy using AAV vectors.

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy directed to the lungs, in particular for treatment of cystic fibrosis (CF). In animal models of lung gene transfer, neutralizing antibodies in serum made in response to vector exposure have been associated with a partial to complete block to repeat transduction by vectors with the same capsid type, thus transduction by AAV vectors might be inefficient in humans previously exposed to the same AAV type. AAV type 2 (AAV2) has been used in clinical trials of lung gene transfer, but AAV5 and AAV6 have been shown to mediate more efficient transduction in rodent lungs and in cultured human airway epithelia compared with that of AAV2. Here we have measured neutralizing antibodies against AAV type 2, 5, and 6 vectors in serum from children and adults with CF, and from normal adults. About 30% of adults were seropositive for AAV2, 20-30% were seropositive for AAV6, and 10-20% were seropositive for AAV5. CF children were seropositive for AAV type 2, 5, or 6 at rates of 4-15%. All individuals seropositive for AAV6 were also seropositive for AAV2, and the AAV6 titer was low compared with the AAV2 titer. AAV5-positive sera were lower both in titers and rates than those seen for AAV6. The results indicate that AAV type 2, 5 or 6 exposure is low in CF and control populations and even lower in CF children.

Adeno-associated virus types 5 and 6 use distinct receptors for cell entry.

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues is determined primarily by the viral capsid proteins. In contrast to vectors made with AAV type 2 capsids, those having type 5 or 6 capsids show high transduction rates in airway epithelial cells, in a range that should be sufficient for treating lung disease. Here we have compared the properties of vectors made with AAV5 or AAV6 capsid proteins to determine whether their receptor usage is similar, and found several differences between the viruses. First, an AAV6 vector did not hemagglutinate red blood cells, whereas an AAV5 vector did, and this property was sialic acid dependent. Second, AAV5 vector transduction required sialic acid in all cells tested, whereas AAV6 vector transduction was sialic acid dependent or independent, depending on the target cells tested. Third, levels of an AAV6 vector that interfered with entry of another AAV6 vector only poorly inhibited AAV5 vector transduction and vice versa. These results indicate that AAV5 and AAV6 vectors use distinct cellular receptors for cell entry. Although both AAV5 and AAV6 vectors exhibited high transduction rates in well-differentiated human airway epithelial cultures, they exhibited distinct cell-type transduction profiles in mouse lung that may reflect differences in receptor usage.

Sheep retrovirus structural protein induces lung tumours.

Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats, with significant animal health and economic consequences. The host range of JSRV is in part limited by species-specific differences in the virus entry receptor, hyaluronidase 2 (Hyal2), which is not functional as a receptor in mice but is functional in humans. Sheep are immunotolerant of JSRV because of the expression of closely related endogenous retroviruses, which are not present in humans and most other species, and this may facilitate oncogenesis. Here we show that expression of the JSRV envelope (Env) protein alone in lungs of mice, by using a replication-incompetent adeno-associated virus vector, results in tumours with a bronchiolo-alveolar localization like those seen in sheep. Whereas lethal disease was observed in immunodeficient mice, tumour development was almost entirely blocked in immunocompetent mice. Our results provide a rare example of an oncogenic viral structural protein, show that interaction of the viral Env protein with the virus entry receptor Hyal2 is not required for tumorigenesis, and indicate that immune recognition of Env can protect against JSRV tumorigenesis.

Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6.

Vectors based on recombinant adeno-associated viruses (rAAV) have emerged as tools of choice for gene transfer to skeletal muscle. rAAV vectors demonstrate efficient, safe, and stable transduction. Multiple serotypes of AAV exist, but vectors based on serotype 2 (rAAV2) are the most thoroughly characterized and frequently employed. Here, we characterize transduction of the skeletal musculature using rAAV vectors pseudotyped with serotype 6 capsid proteins (rAAV6). We demonstrate that rAAV6 vectors can efficiently transduce the skeletal musculature of mice at levels >500-fold higher than is achievable with rAAV2 vectors and can readily saturate individual muscles following direct injection. Further, rAAV6 vectors are capable of transducing the diaphragm and intercostal muscles of mice after a simple injection into the intrathoracic cavity and are capable of widespread transduction throughout the musculature of mice injected in the intraperitoneal space as newborn pups. These results demonstrate that rAAV6 vectors hold great potential for use in gene delivery protocols targeting the skeletal musculature.

AAV-mediated gene transfer to mouse lungs.

The ability of adeno-associated viral (AAV) vectors to promote persistent gene expression in nondividing cells in multiple somatic tissues of animals (1-4) makes them excellent tools for gene transfer. One tissue of interest for gene transfer is the lung epithelium, which is afflicted in cystic fibrosis (CF). However, although initial animal studies done with vectors based on AAV type 2 have demonstrated transduction in multiple cells types in the lung, the rates were modest in alveolar cells and much lower rates in airway epitheila and required high particle numbers (5-7). In contrast, an AAV6 encapsidated vector showed preferential transduction of epithelial cells in large and small airways (8) at rates that exceeded the 5% efficiency rate predicted to have a therapeutic value for CF gene therapy (9). In fact, recent studies using vectors based on other AAV types showed that types 1-6 have different tissue tropisms (10-15), and that types 5 and 6 are more efficient than type 2 in lung epithelium (8,14). In mouse lung, an AAV2 vector gave modest transduction rates.

Jaagsiekte sheep retrovirus envelope efficiently pseudotypes human immunodeficiency virus type 1-based lentiviral vectors.

Jaagsiekte sheep retrovirus (JSRV) infects lung epithelial cells in sheep, and oncoretroviral vectors bearing JSRV Env can mediate transduction of human cells, suggesting that such vectors might be useful for lung-directed gene therapy. Here we show that JSRV Env can also efficiently pseudotype a human immunodeficiency virus type 1-based lentiviral vector, a more suitable vector for transduction of slowly dividing lung epithelial cells. We created several chimeric Env proteins that, unlike the parental Env, do not transform rodent fibroblasts but are still capable of pseudotyping lentiviral and oncoretroviral vectors.

Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene.

The small packaging capacity of adeno-associated virus (AAV) vectors limits the utility of this promising vector system for transfer of large genes. We explored the possibility that larger genes could be reconstituted following homologous recombination between AAV vectors carrying overlapping gene fragments. An alkaline phosphatase (AP) gene was split between two such AAV vectors (rec vectors) and packaged using AAV2 or AAV6 capsid proteins. Rec vectors having either capsid protein recombined to express AP in cultured cells at about 1-2% of the rate observed for an intact vector. Surprisingly, the AAV6 rec vectors transduced lung cells in mice almost as efficiently as did an intact vector, with 10% of airway epithelial cells, the target for treatment of cystic fibrosis (CF), being positive. Thus AAV rec vectors may be useful for diseases such as CF that require transfer of large genes.