Infectivity of adeno-associated virus serotypes in mouse testis

Background

Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection.

Results

We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells.

Conclusions

In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.

     

Pseudotyped AAV Vector-Mediated Gene Transfer in a Human Fetal Trachea Xenograft Model: Implications for In Utero Gene Therapy for Cystic Fibrosis

Background

Lung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls.

Methodology/Principal Findings

Human fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts.

Conclusions/Significance

Based on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis.     

Aromatase and 5-alpha reductase gene expression: modulation by pain and morphine treatment in male rats

Background

Neuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.

Results

Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP.

Conclusions

The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.     

GFAP-Driven GFP Expression in Activated Mouse Müller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors

Background

Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.

Methodology/Principal Findings

We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1^−/− retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.

Conclusions/Significance

Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.     

Ocular Localization and Transduction by Adenoviral Vectors Are Serotype-Dependent and Can Be Modified by Inclusion of RGD Fiber Modifications

Purpose

To evaluate localization and transgene expression from adenoviral vector of serotypes 5, 35, and 28, ± an RGD motif in the fiber following intravitreal or subretinal administration.

Methods

Ocular transduction by adenoviral vector serotypes ± RGD was studied in the eyes of mice receiving an intravitreous or subretinal injection. Each serotype expressed a CMV-GFP expression cassette and histological sections of eyes were examined. Transgene expression levels were examined using luciferase (Luc) regulated by the CMV promoter.

Results

GFP localization studies revealed that serotypes 5 and 28 given intravitreously transduced corneal endothelial, trabecular, and iris cells. Intravitreous delivery of the unmodified Ad35 serotype transduced only trabecular meshwork cells, but, the modification of the RGD motif into the fiber of the Ad35 viral vector base expanded transduction to corneal endothelial and iris cells. Incorporation of the RGD motif into the fiber knob with deletion of RGD from the penton base did not affect the transduction ability of the Ad5 vector base. Subretinal studies showed that RGD in the Ad5 knob shifted transduction from RPE cells to photoreceptor cells. Using a CMV-Luc expression cassette, intravitreous delivery of all the tested vectors, such as Ad5-, Ad35- and Ad28- resulted in an initial rapid induction of luciferase activity that thereafter declined. Subretinal administration of vectors showed a marked difference in transgene activity. Ad35-Luc gene expression peaked at 7 days and remained elevated for 6 months. Ad28-Luc expression was high after 1 day and remained sustained for one month.

Conclusions

Different adenoviral vector serotypes ± modifications transduce different cells within the eye. Transgene expression can be brief or extended and is serotype and delivery route dependent. Thus, adenoviral vectors provide a versatile platform for the delivery of therapeutic agents for ocular diseases.     

Impact of transduction towards the proliferation and migration as well as the transduction efficiency of human umbilical cord-derived late endothelial progenitor cells with nine recombinant adeno-associated virus serotypes

Objectives

To evaluate the transduction efficiency of human umbilical cord-derived, late endothelial progenitor cells late (HUCB-late EPCs) with nine recombinant adeno-associated virus (rAAV) serotypes and the ability of proliferation and migration of the cells after transduction.

Results

rAAV2 and rAAV6 showed a greater ability than other serotypes to transduce late EPCs (P < 0.05). After transduction, cell proliferation ability weakened (P < 0.05), but the ability of migration to stromal cell-derived factor (SDF-1) unchanged.

Conclusion

There is an advantage of choosing the optimal rAAV serotype as a gene vector to alter the biologic characteristics of late EPCs.

     

The effect of DNA-dependent protein kinase on adeno-associated virus replication

Background

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme and plays an important role in determining the molecular fate of the rAAV genome. However, the effect this cellular enzyme on rAAV DNA replication remains elusive.

Methodology/Principal Findings

In the present study, we characterized the roles of DNA-PK on recombinant adeno-associated virus DNA replication. Inhibition of DNA-PK by a DNA-PK inhibitor or siRNA targeting DNA-PKcs significantly decreased replication of AAV in MO59K and 293 cells. Southern blot analysis showed that replicated rAAV DNA formed head-to-head or tail-to-tail junctions. The head-to-tail junction was low or undetectable suggesting AAV-ITR self-priming is the major mechanism for rAAV DNA replication. In an in vitro replication assay, anti-Ku80 antibody strongly inhibited rAAV replication, while anti-Ku70 antibody moderately decreased rAAV replication. Similarly, when Ku heterodimer (Ku70/80) was depleted, less replicated rAAV DNA were detected. Finally, we showed that AAV-ITRs directly interacted with Ku proteins.

Conclusion/Significance

Collectively, our results showed that that DNA-PK enhances rAAV replication through the interaction of Ku proteins and AAV-ITRs.     

Polymers for improving the in vivo transduction efficiency of AAV2 vectors

Background

Adeno-associated virus has attracted great attention as vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy infusion of very large amounts of vectors is required. This not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome these problems would be to find strategies able to increase the in vivo efficiency.

Methodology

Here, we investigated whether polymers can act as adjuvants to increase the in vivo efficiency of AAV2. Our strategy consisted in the pre-injection of polymers before intravenous administration of mice with AAV2 encoding a murine secreted alkaline phosphatase (mSeAP). The transgene expression, vector biodistribution and tissue transduction were studied by quantification of the mSeAP protein and real time PCR. The injection of polyinosinic acid and polylysine resulted in an increase of plasmatic mSeAP of 2- and 12-fold, respectively. Interestingly, polyinosinic acid pre-injection significantly reduced the neutralizing antibody titer raised against AAV2.

Conclusions

Our results show that the pre-injection of polymers can improve the overall transduction efficiency of systemically administered AAV2 and reduce the humoral response against the capsid proteins.     

Creating a Novel Origin of Replication through Modulating DNA-Protein Interfaces

Background

While the molecular mechanisms of DNA-protein specificity at the origin of replication have been determined in many model organisms, these interactions remain unknown in the majority of higher eukaryotes and numerous vertebrate viruses. Similar to many viral origins of replication, adeno-associated virus (AAV) utilizes a cis-acting origin of replication and a virus specific Replication protein (Rep) to faithfully carry out self-priming replication. The mechanisms of AAV DNA replication are generally well understood. However, the molecular basis of specificity between the Rep protein and the viral origin of replication between different AAV serotypes remains uncharacterized.

Methodology/Principal Findings

By generating a panel of chimeric and mutant origins between two AAV serotypes, we have mapped two independent DNA-Protein interfaces involved in replicative specificity. In vivo replication assays and structural modeling demonstrated that three residues in the AAV2 Rep active site are necessary to cleave its cognate origin. An analogous origin (AAV5) possesses a unique interaction between an extended Rep binding element and a 49 aa region of Rep containing two DNA binding interfaces.

Conclusions/Significance

The elucidation of these structure-function relationships at the AAV origin led to the creation of a unique recombinant origin and compatible Rep protein with properties independent of either parent serotype. This novel origin may impact the safety and efficacy of AAV as a gene delivery tool. This work may also explain the unique ability of certain AAV serotypes to achieve site-directed integration into the human chromosome. Finally, this result impacts the study of conserved DNA viruses which employ rolling circle mechanisms of replication.     

MicroRNA-restricted transgene expression in the retina

Background

Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions.

Methodology/Principal Findings

To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3′UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy.

Conclusions

We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters.     

A Novel Adeno-Associated Viral Variant for Efficient and Selective Intravitreal Transduction of Rat Müller Cells

Background

The pathologies of numerous retinal degenerative diseases can be attributed to a multitude of genetic factors, and individualized treatment options for afflicted patients are limited and cost-inefficient. In light of the shared neurodegenerative phenotype among these disorders, a safe and broad-based neuroprotective approach would be desirable to overcome these obstacles. As a result, gene delivery of secretable-neuroprotective factors to Müller cells, a type of retinal glia that contacts all classes of retinal neurons, represents an ideal approach to mediate protection of the entire retina through a simple and innocuous intraocular, or intravitreal, injection of an efficient vehicle such as an adeno-associated viral vector (AAV). Although several naturally occurring AAV variants have been isolated with a variety of tropisms, or cellular specificities, these vectors inefficiently infect Müller cells via intravitreal injection.

Methodology/Principal Findings

We have previously applied directed evolution to create several novel AAV variants capable of efficient infection of both rat and human astrocytes through iterative selection of a panel of highly diverse AAV libraries. Here, in vivo and in vitro characterization of these isolated variants identifies a previously unreported AAV variant ShH10, closely related to AAV serotype 6 (AAV6), capable of efficient, selective Müller cell infection through intravitreal injection. Importantly, this new variant shows significantly improved transduction relative to AAV2 (>60%) and AAV6.

Conclusions/Significance

Our findings demonstrate that AAV is a highly versatile vector capable of powerful shifts in tropism from minor sequence changes. This isolated variant represents a new therapeutic vector to treat retinal degenerative diseases through secretion of neuroprotective factors from Müller cells as well as provides new opportunities to study their biological functions in the retina.     

Unique biologic properties of recombinant AAV1 transduction in polarized human airway epithelia

The choice of adeno-associated virus serotypes for clinical applications is influenced by the animal model and model system used to evaluate various serotypes. In the present study, we sought to compare the biologic properties of rAAV2/1, rAAV2/2, and rAAV2/5 transduction in polarized human airway epithelia using viruses purified by a newly developed common column chromatography method. Results demonstrated that apical transduction of human airway epithelia with rAAV2/1 was 100-fold more efficient than rAAV2/2 and rAAV2/5. This transduction profile in human airway epithelia (rAAV2/1 > rAAV2/2 = rAAV2/5) was significantly different from that seen following nasal administration of these vectors to mouse lung (rAAV2/5 > rAAV2/1 > rAAV2/2), emphasizing differences in transduction of these serotypes between these two species. In stark contrast to rAAV2/2 and rAAV2/5, rAAV2/1 transduced both the apical and basolateral membrane of human airway epithelia with similar efficiency. However, the overall level of transduction across serotypes did not correlate with vector internalization. We hypothesized that differences in post-entry processing of these serotypes might influence the efficiency of apical transduction. To this end, we tested the effectiveness of proteasome inhibitors to augment nuclear translocation and gene expression from the three serotypes. Augmentation of rAAV2/1 apical transduction of human polarized airway epithelia was 10-fold lower than that for rAAV2/2 and rAAV2/5. Cellular fractionation studies demonstrated that proteasome inhibitors more significantly enhanced rAAV2/2 and rAAV2/5 translocation to the nucleus than rAAV2/1. These results demonstrate that AAV1 transduction biology in human airway epithelia differs from that of AAV2 and AAV5 by virtue of altered ubiquitin/proteasome sensitivities that influence nuclear translocation.     

Ab-externo AAV-mediated gene delivery to the suprachoroidal space using a 250 micron flexible microcatheter

Background

The current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV) vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter.

Methods

6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP.

Results

Immunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically.

Conclusions

The ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery.     

DupChecker: a bioconductor package for checking high-throughput genomic data redundancy in meta-analysis

Background

Meta-analysis has become a popular approach for high-throughput genomic data analysis because it often can significantly increase power to detect biological signals or patterns in datasets. However, when using public-available databases for meta-analysis, duplication of samples is an often encountered problem, especially for gene expression data. Not removing duplicates could lead false positive finding, misleading clustering pattern or model over-fitting issue, etc in the subsequent data analysis.

Results

We developed a Bioconductor package Dupchecker that efficiently identifies duplicated samples by generating MD5 fingerprints for raw data. A real data example was demonstrated to show the usage and output of the package.

Conclusions

Researchers may not pay enough attention to checking and removing duplicated samples, and then data contamination could make the results or conclusions from meta-analysis questionable. We suggest applying DupChecker to examine all gene expression data sets before any data analysis step.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-323) contains supplementary material, which is available to authorized users.     

The treatment of glioblastoma multiforme through activation of microglia and TRAIL induced by rAAV2-mediated IL-12 in a syngeneic rat model

Background

Microglial cells are the predominant immune cells in malignant brain tumors, but tumors may release some factors to reduce their defensive functions. Restoration of the anti-cancer function of microglia has been proposed as a treatment modality for glioblastoma. We examined the effect of intra-cranially administered recombinant adeno-associated virus encoding interleukin-12 (rAAV2/IL12) on transfection efficiency, local immune activity and survival in a rat model of glioblastoma multiforme.

Methods

F344 rats were injected with rAAV2/IL12 and implanted with syngeneic RG2 cells (glioblastoma cell line). Intracerebral interleukin-12 and interferon-γ concentrations were determined by ELISA. Activation of microglia was determined by expressions of ED1 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) which were evaluated by Western blotting and immunohistochemistry. The proliferation of cancer cells was evaluated with Ki67 immunohistochemistry and apoptosis of cancer cells with TUNEL.

Results

The brains treated with rAAV2/IL-12 maintained high expression of interleukin-12 and interferon-γ for at least two months. In syngeneic tumor model, brains treated with rAAV2/IL12 exhibited more infiltration of activated microglia cells as examined by ED1 and TRAIL stains in the tumor. In addition, the volume of tumor was markedly smaller in AAV2/IL12-treated group and the survival time was significantly longer in this group too.

Conclusion

The intra-cerebrally administered rAAV2/IL-12 efficiently induces long lasting expression of IL-12, the greater infiltration of activated microglia cells in the tumor associated improved immune reactions, resulting in the inhibited growth of implanted glioblastoma and the increased survival time of these rats.     

Axonal-Transport-Mediated Gene Transduction in the Interior of Rat Bone

Background

Gene transduction has been considered advantageous for the sustained delivery of proteins to specific target tissues. However, in the case of hard tissues, such as bone, local gene delivery remains problematic owing to anatomical accessibility limitations of the target sites.

Methodology/Principal Findings

Here, we evaluated the feasibility of exogenous gene transduction in the interior of bone via axonal transport following intramuscular administration of a nonviral vector. A high expression level of the transduced gene was achieved in the tibia ipsilateral to the injected tibialis anterior muscle, as well as in the ipsilateral sciatic nerve and dorsal root ganglia. In sciatic transection rats, the gene expression level was significantly lowered in bone.

Conclusions/Significance

These results suggest that axonal transport is critical for gene transduction. Our study may provide a basis for developing therapeutic methods for efficient gene delivery into hard tissues.     

Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders

Background

Adeno-associated virus (AAV) is well established as a vehicle for in vivo gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for in vivo functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail.

Methodology/Principal Findings

We subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing Crx under the control of the Crx 2kb promoter into the neonatal Crx KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the Crx KO retina.

Conclusion/Significance

In the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of Crx.     

Adeno-associated virus type 12 (AAV12): a novel AAV serotype with sialic acid- and heparan sulfate proteoglycan-independent transduction activity

Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy. Recent isolations of novel AAV serotypes have led to significant advances by broadening the tropism and increasing the efficiency of gene transfer to the desired target cell. However, a major concern that remains is the strong preexisting immune responses to several vectors. In this paper, we describe the isolation and characterization of AAV12, an AAV serotype with unique biological and immunological properties. In contrast to those of all other reported AAVs, AAV12 cell attachment and transduction do not require cell surface sialic acids or heparan sulfate proteoglycans. Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. The feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications.