Development Of Adeno-associated Virus Vectors Research Articles

In vivo Delivery Tools for Clustered Regularly Interspaced Short Palindromic Repeat/Associated Protein 9-Mediated Inhibition of Hepatitis B Virus Infection: An Update.

Chronic hepatitis B virus (HBV) infection remains a major global health problem despite the availability of an effective prophylactic HBV vaccine. Current antiviral therapies are unable to fully cure chronic hepatitis B (CHB) because of the persistent nature of covalently closed circular DNA (cccDNA), a replicative template for HBV, which necessitates the development of alternative therapeutic approaches. The CRISPR/Cas system, a newly emerging genome editing tool, holds great promise for genome editing and gene therapy. Several in vitro and/or in vivo studies have demonstrated the effectiveness of HBV-specific clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) systems in cleaving HBV DNA and cccDNA. Although recent advances in CRISPR/Cas technology enhance its prospects for clinical application against HBV infection, in vivo delivery of the CRISPR/Cas9 system at targets sites remains a major challenge that needs to be resolved before its clinical application in gene therapy for CHB. In the present review, we discuss CRISPR/Cas9 delivery tools for targeting HBV infection, with a focus on the development of adeno-associated virus vectors and lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery to treat CHB. In addition, we discuss the importance of delivery tools in the enhancement of the antiviral efficacy of CRISPR/Cas9 against HBV infection.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response.

A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.

Adeno‐associated virus‐vectored influenza vaccine elicits neutralizing and Fcγ receptor‐activating antibodies

The current seasonal inactivated influenza vaccine protects only against a narrow range of virus strains as it triggers a dominant antibody response toward the hypervariable hemagglutinin (HA) head region. The discovery of rare broadly protective antibodies against conserved regions in influenza virus proteins has propelled research on distinct antigens and delivery methods to efficiently induce broad immunity toward drifted or shifted virus strains. Here, we report that adeno‐associated virus (AAV) vectors expressing influenza virus HA or chimeric HA protected mice against homologous and heterologous virus challenges. Unexpectedly, immunization even with wild‐type HA induced antibodies recognizing the HA‐stalk and activating FcγR‐dependent responses indicating that AAV‐vectored expression balances HA head‐ and HA stalk‐specific humoral responses. Immunization with AAV‐HA partially protected also ferrets against a harsh virus challenge. Results from this study provide a rationale for further clinical development of AAV vectors as influenza vaccine platform, which could benefit from their approved use in human gene therapy.

Humoral Immune Response to AAV

Adeno-associated virus (AAV) is a member of the family Parvoviridae that has been widely used as a vector for gene therapy because of its safety profile, its ability to transduce both dividing and non-dividing cells, and its low immunogenicity. AAV has been detected in many different tissues of several animal species but has not been associated with any disease. As a result of natural infections, antibodies to AAV can be found in many animals including humans. It has been shown that pre-existing AAV antibodies can modulate the safety and efficacy of AAV vector-mediated gene therapy by blocking vector transduction or by redirecting distribution of AAV vectors to tissues other than the target organ. This review will summarize antibody responses against natural AAV infections, as well as AAV gene therapy vectors and their impact in the clinical development of AAV vectors for gene therapy. We will also review and discuss the various methods used for AAV antibody detection and strategies to overcome neutralizing antibodies in AAV-mediated gene therapy.

Novel adeno-associated viral vectors for retinal gene therapy

Vectors derived from adeno-associated virus (AAV) are currently the most promising vehicles for therapeutic gene delivery to the retina. Recently, subretinal administration of AAV2 has been demonstrated to be safe and effective in patients with a rare form of inherited childhood blindness, suggesting that AAV-mediated retinal gene therapy may be successfully extended to other blinding conditions. This is further supported by the great versatility of AAV as a vector platform as there are a large number of AAV variants and many of these have unique transduction characteristics useful for targeting different cell types in the retina including glia, epithelium and many types of neurons. Naturally occurring, rationally designed or in vitro evolved AAV vectors are currently being utilized to transduce several different cell types in the retina and to treat a variety of animal models of retinal disease. The continuous and creative development of AAV vectors provides opportunities to overcome existing challenges in retinal gene therapy such as efficient transfer of genes exceeding AAV's cargo capacity, or the targeting of specific cells within the retina or transduction of photoreceptors following routinely used intravitreal injections. Such developments should ultimately advance the treatment of a wide range of blinding retinal conditions.

CLINICAL DEVELOPMENT OF AAV VECTORS

Impressive progress has been made in clinical development of adeno-associated virus (AAV) vectors over the last 15 years in more than 40 clinical trials, involving many hundreds of subjects with vector delivery by many different routes and at a wide range of doses. Preclinical expectations, including an excellent safety profile, the tendency to remain at a local delivery site, and the persistence of expression for years after delivery, are being borne out in clinical trials. Immune responses to the vectors in human subjects, less well anticipated by preclinical studies, are now being examined in current AAV vector clinical trials, in order to determine in what circumstances such responses might occur or be problematic. Development of AAV vectors is likely to continue to be an expanding field. The current successes in the subjects with retinal degeneration and Parkinson's disease are highly encouraging for AAV vectors and for the whole field of gene therapy.

Evidence for the Failure of Adeno-associated Virus Serotype 5 to Package a Viral Genome ≥8.2 kb

Limited packaging capacity hinders adeno-associated virus (AAV) gene therapy. A recent study seems to have provided a solution to this problem. Allocca et al. reported that AAV-5 could package an 8.9 kb vector genome. Here we tested whether this approach can be used to deliver a large genome for Duchenne muscular dystrophy (DMD) gene therapy. We first evaluated AAV-5 packaging of an 8.2 kb genome. This vector carries two independent reporter gene cassettes, one for alkaline phosphatase (AP) and another for LacZ. Viral yield was log-fold lower than that of a regular AAV-5. Nevertheless, both AP and LacZ genes were detected in purified virus. Injection to dystrophic muscle resulted in both AP and LacZ expression. On electron microscopy, virion structure appeared normal. Surprisingly, we did not find the full-length single-stranded viral genome by alkaline gel electrophoresis. Neither did we see the full-length double-stranded replication forms in adenovirus coinfected cells. We suspect that AP and LacZ expression may have come from partially packaged 5' or 3'-half of the genome. Additional studies revealed failure of AAV-5 to package and express an 8.7 kb minidystrophin gene cassette. In summary, our results do not support the extraordinary packaging capacity of AAV-5.

Transient transfection methods for clinical adeno-associated viral vector production.

Recombinant adeno-associated virus (AAV)-based vectors expressing therapeutic gene products have shown great potential for human gene therapy. One major challenge for translation of promising research to clinical development is the manufacture of sufficient quantities of AAV vectors that meet stringent standards for purity, potency, and safety required for human parenteral administration. Several methods have been developed to generate recombinant AAV in cell culture, each offering distinct advantages. Transient transfection-based methods for vector production are reviewed here, with a focus on specific considerations for development of AAV vectors as clinical products.

Adeno-associated Virus of a Single-polarity DNA Genome Is Capable of Transduction In Vivo

The adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. AAV vectors with wild-type inverted terminal repeats package either the plus- or the minus-strand DNA genomes with equal frequency. By creating a series of deletions within the, we have developed a genetic approach that can generate an AAV vector that packages its single-stranded DNA genome predominantly in a single polarity (99.4%). This novel reagent efficiently transduced muscle, brain and liver in whole animals. The transduction efficiencies were similar to those of the control mixed-polarity vectors. Our results showed that reannealing of plus- and minus-strand DNA was not required for AAV-mediated transduction in vivo, supporting the hypothesis that second-strand DNA synthesis is a primary pathway in converting the single-stranded AAV genome into double-stranded forms. The availability of the single-polarity AAV vector would aid further studies on the mechanism of AAV transduction as well as the application of AAV vector for gene replacement therapy.

Efficient Whole-body Transduction with Trans-splicing Adeno-associated Viral Vectors

Limited packaging capacity has hampered adeno-associated virus (AAV)-mediated gene therapy for many common genetic diseases such as cystic fibrosis (CF) and Duchenne muscular dystrophy (DMD). Trans-splicing AAV (tsAAV) vectors double AAV packaging capacity but their transduction efficiency has been too low to be useful. We have recently overcome this hurdle by rational vector design. We have shown that a pair of optimized mini-dystrophin tsAAV vectors can reach the same transduction efficiency as that of a single AAV vector after local injection in dystrophic muscle. However, global gene transfer is required to treat diseases like DMD. To test whether systemic delivery can be achieved with tsAAV vectors, we generated a set of optimized alkaline phosphatase (AP) tsAAV vectors. We delivered AAV serotype 9 pseudotyped AP tsAAV intravenously to newborn mice. Six weeks later, we observed high-level transduction in all body skeletal muscle and the heart, the tissues that are affected in DMD. We also detected efficient transduction in the lung, the primary organ affected in CF. Our results provide the first evidence of whole-body transduction with tsAAV vectors and further raise the hope of tsAAV gene therapy for DMD and CF.

848. Development of AAV Vectors Targeted to Vascular Endothelium

Top of pageAbstract Many diseases, such as cancer and forms of cardiovascular disease, share a common process of vascular proliferation. It is thought that the ability to deliver and express genes in the vasculature will be valuable for the development of gene therapies aimed at these diseases. While adeno-associated virus (AAV) vectors can transduce a wide variety of cell types, certain cells (including cells of the vascular system) remain refractive to AAV transduction. We have pursued several different approaches to develop AAV vectors capable of gene delivery to the vasculature. Peptides, targeted towards vascular endothelial growth factor receptor 2 (VEGFR2) or integrins and peptides homologous to the N-terminal fragment of human high mobility group protein 2 (HMGN2), were incorporated into the capsids of AAV vectors. Two different sites were utilized for peptide insertion. The first site was designed to display inserted peptides on all viral capsid proteins (VP1, VP2, and VP3), while the second site was chosen to display peptides only on the VP2 capsid protein. Incorporating peptides into all capsid proteins allows for a greater number of the peptides to be displayed, but the size of the peptide was limited to |[le]| 24 amino acids. While the number of peptides displayed following VP2 insertion is limited (|[sim]|3 per viral particle), this site allows for the display of a much larger peptides while maintaining vector infectivity. Near wild type titers were obtained for all modified vectors. We also generated AAV2 vectors unable to bind their natural receptor, heparan sulfate proteoglycan (HSPG), by incorporating two point mutations into the VP3 coding region of the AAV2 genome (R585A/R588A). A solid-phase heparin sulfate binding assay was developed and used to show that these HS-ablated AAV2 vectors, as well as the HS-ablated AAV2 vectors containing targeting ligand insertions were unable to bind HS. Targeting insertions were made in AAV1, AAV2, and HS-ablated AAV2 backbones with the idea that HS-ablated AAV2 vectors might allow for the highest specificity, while targeted AAV1 vectors might allow for the highest level of in vivo vascular transduction due to the greater in vivo gene transfer observed with unmodified AAV1 vectors compared to AAV2 vectors. Along with the targeting peptide, a biotin acceptor peptide (BAP), used to purify modified AAV1 and HS-ablated AAV2 vectors, was incorporated into some modified AAV capsids. Vectors were used to infect four different human endothelial cell populations (saphenous vein endothelial cells (HSaVEC), umbilical vein endothelial cells (HUVEC), coronary artery endothelial cells (HCAEC), and coronary microvascular endothelial cells (HCMVEC)). Integrin targeted AAV1 and the integrin targeted/BAP chimeric AAV1 vectors displayed significantly higher levels of gene transfer to all endothelial cell types compared to unmodified AAV1 vector. Transduction of HSaVECs was greater than 20 fold (p<0.002) more efficient with the RGD/BAP chimeric AAV1 than with untargeted AAV1. Similarly, in HCAECs, AAV1-RGD/BAP was greater than 65 fold (p<0.0001) more efficient, and AAV1-VEGFR2E/BAP was 4 fold (p=0.044) more efficient than untargeted vectors.

Adeno-associated virus and the development of adeno-associated virus vectors: a historical perspective

Adeno-associated virus and the development of adeno-associated virus vectors: a historical perspective

Development of adeno-associated virus vectors for gene therapy of cystic fibrosis.

The potential application of adeno-associated virus (AAV) as a gene delivery system in mammalian cells was originally indicated by several features of its life cycle. Most importantly, in the absence of a helper virus for productive AAV replication, the AAV genome can persist in host cells and this may involve integration into the host cell genome. Appreciation of the technical feasibility of this potential gene therapy application arose from cloning infectious AAV genomes (Samulski et al. 1982; Laughlin et al. 1983). The earliest AAV vectors were reported about 10 years ago (Tratschin et al. 1984; Hermonat and Muzyczka 1984) but progress from there was slow because very few laboratories were engaged in this work. However, recent developments have highlighted the potential use of AAV vectors for gene therapy (Flotte et al. 1993a, b; Kaplitt et al. 1994) and AAV vectors are now being developed for imminent clinical trials in patients with cystic fibrosis (Flotte et al. 1993b, 1994a).