Kenneth I. Berns

Gene Therapy Using Adeno-Associated Virus Vectors

SUMMARY The unique life cycle of adeno-associated virus (AAV) and its ability to infect both nondividing and dividing cells with persistent expression have made it an attractive vector. An additional attractive feature of the wild-type virus is the lack of apparent pathogenicity. Gene transfer studies using AAV have shown significant progress at the level of animal models; clinical trials have been noteworthy with respect to the safety of AAV vectors. No proven efficacy has been observed, although in some instances, there have been promising observations. In this review, topics in AAV biology are supplemented with a section on AAV clinical trials with emphasis on the need for a deeper understanding of AAV biology and the development of efficient AAV vectors. In addition, several novel approaches and recent findings that promise to expand AAVs utility are discussed, especially in the context of combining gene therapy ex vivo with new advances in stem or progenitor cell biology.

Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination.

The human parvovirus, adeno‐associated virus (AAV), has been shown to integrate preferentially into human chromosome 19 q13.3‐qter. The human target sequence for AAV integration (AAVS1) was cloned and sequenced. By analysis of the proviral junctions it was determined that integration of the AAV DNA occurred via a non‐homologous recombination pathway although there were either four or five identical nucleotides at the junctions. Integration was a multistep, concerted process that resulted in cellular sequence rearrangements. The sequence of the integration locus was analyzed for possible recombination signals. Direct repeats at a much greater than random occurrence were found distributed non‐uniformly throughout the AAVS1 sequence. A CpG island containing transcription factor binding site elements is suggestive of a TATA‐less promoter. Evidence for transcriptional activity was provided by PCR amplification of reverse transcribed RNA.

Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter

A human parvovirus, adeno-associated virus (AAV), is unique among eukaryotic DNA viruses in its ability to integrate site specifically into a defined region of human chromosome 19. In this study we used in situ hybridization to visualized directly the site of AAV DNA integration in latently infected human cell lines and normal human cells.

Biology of Adeno-associated Virus

Adeno-associated Virus (AAV) is classified as a member of the family Parvoviridae (Siegl et al. 1985; Berns 1990a). Members are small, nonenveloped, icosahedral viruses (diameter ca. 20–26 nm) with linear, single-stranded DNA genomes of 4.7–6 kb. Parvoviridae have been isolated from many species ranging from insects to humans. AAV is assigned to the genus Dependovirus, so named because the virus was discovered as a contaminant in purified adenovirus (Ad) stocks and in most instances does not productively infect cells in culture unless there is a coinfection by an unrelated helper virus, which is most commonly Ad, but also can be any type of herpesvirus (Atchinson et al. 1965; Hoggan et al. 1966; Buller et al. 1981). Various serotypes have been isolated from birds and many mammalian species, including humans. About 90% of U.S. adults are seropositive, but in no case has the virus been implicated as the etiological agent for a human disease or as the cause of disease in any other species. Because of the requirement for a helper coinfection for productive infection in cell culture, AAV was long considered to be a defective virus. Detailed studies described below have demonstrated that the virus is not defective, but rather preferentially establishes a latent infection in a healthy cell and is only induced to undergo productive vegetative multiplication when the host cell is stressed.

Rescue of adeno-associated virus from recombinant plasmids: Gene correction within the terminal repeats of AAV

We have isolated three types of pBR322-AAV recombinant plasmids that contain deletions within the 145 bp AAV terminal repeats. When the plasmids were transfected into human cells, mutants that contained deletions within the left (type I) or right (type II) terminal repeat were viable. Of four mutants examined that contained deletions in both termini (type III), only one was viable. All of the viable mutants produced AAV virions that contained wild-type AAV DNA. Furthermore, the viable type III deletion could be converted to a nonviable mutant by deleting all copies of an 11 bp sequence from its termini. We conclude that there is an efficient mechanism for correcting deletions within the AAV termini. A model that could account for these observations is also discussed.

Adeno-Associated Viruses: An Update

Publisher Summary The adeno-associated viruses (AAV) are the only known DNA animal viruses that are absolutely dependent upon coinfection by a second unrelated virus to undergo productive infection. They are members of the family Parvoviridae , which are among the smallest of the DNA animal viruses. The Parvoviridae genome is a linear single-stranded DNA molecule approximately 5 kb in size, which is encapsidated in a naked icosahedral particle 18–27 nm in diameter. In addition to one genus of insect viruses (densoviruses), the family contains two genera that infect a broad spectrum of vertebrates ranging from birds to humans. The parvoviruses are able to replicate autonomously in infected cells but require actively dividing cells for a productive infection. Although the dependoviruses (AAV) are structurally similar to the autonomous parvoviruses, they are absolutely defective and require coinfection with structurally unrelated adenoviruses or herpesviruses for a productive infection to occur. Adeno-associated virus does not have any structural relatedness to either of its helpers; on the other hand, the three viruses do represent all of the known vertebrate virus families with linear DNA genomes that replicate in cell nuclei.

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA☆

Abstract The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10 6 . Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.

Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors.

Previous work from our group showed that recombinant adeno-associated virus (rAAV) vectors mediated long-term secretion of therapeutic serum levels of human alpha-1 antitrypsin (hAAT) after a single injection in murine muscle. We hypothesized that hepatocyte transduction could be even more efficient, since these cells represent the natural site of AAT production and secretion. To test this hypothesis, rAAV vectors containing the hAAT cDNA driven by either the human elongation factor 1 alpha promoter, the human cytomegalovirus immediate–early promoter (CMV), or the CMV-chicken beta actin hybrid (CB) promoter were injected into the portal or tail veins of adult C57Bl/6 mice. Potentially therapeutic serum levels of hAAT (600 μg/ml) were achieved after portal vein injection of doses of 4 × 109 infectious units (IU), a 10-fold lower dose than that required for similar levels of expression via the i.m. route. Serum levels greater than 1 mg/ml were achieved at doses of 3 × 1010 IU. Southern blotting of liver DNA revealed the presence of circular episomal vector genomes. Immunostaining showed that transgene expression was scattered throughout the liver parenchyma. Similar results were obtained with a rAAV-CB-green fluorescent protein (GFP) vector. There was no evidence of hepatic toxicity. These data indicate that liver-directed rAAV-based gene therapy is effective in the murine model, and hence might be feasible for treatment of human AAT deficiency.

Adeno-associated virus type-2 expression of pigmented epithelium-derived factor or Kringles 1–3 of angiostatin reduce retinal neovascularization

Neovascular diseases of the retina include age-related macular degeneration and diabetic retinopathy, and together they comprise the leading causes of adult-onset blindness in developed countries. Current surgical, pharmaceutical, and laser therapies for age-related macular degeneration (AMD) rarely result in improved vision, do not significantly prevent neovascularization (NV), and often result in at least some vision loss. To address this therapeutic gap, we determined the efficacy of recombinant adeno-associated viral (rAAV) serotype-2-mediated expression of pigment epithelium-derived factor (PEDF) or Kringle domains 1–3 of angiostatin (K1K3) in reducing aberrant vessel formation in a mouse model of ischemia-induced retinal NV. Both PEDF and K1K3 are potent inhibitors of NV when injected directly, hence expression of these therapeutic factors from rAAV may provide long-term protection from neovascular eye disease. rAAV vectors expressing the therapeutic gene were injected into one eye of postnatal day 0 (P0) newborn mouse pups. Retinal NV was induced in P7 mice by exposure to elevated oxygen for 5 days followed by room air for another five days. Retinal NV was quantified by the number of vascular-endothelial-cell nuclei above the inner-limiting membrane in P17 eyes. The number of such vascular endothelial cell nuclei in eyes treated with rAAV-PEDF or rAAV-K1K3 was significantly reduced (both P < 0.0000002) compared with control eyes. Ocular protein levels detected by ELISA correlate well with the reduction in NV and confirm that expression of antineovascular agents from rAAV vectors may be a therapeutically useful treatment of retinal or choroidal neovascular disease.

Organization of adeno-associated virus DNA in latently infected detroit 6 cells

The DNA of a human dependovirus, adeno-associated virus (AAV), integrates into the cellular genome under conditions nonpermissive for viral DNA replication. The AAV DNA can be rescued from the cellular genome by superinfection with a helper virus, e.g., adenovirus. To characterize the organization of the proviral DNA in greater detail, we isolated three proviral subfragments from a latently infected human cell line. Our findings, based on sequence analysis, restriction enzyme mapping, and genomic blots, demonstrate that the viral terminal repeats (trs) are present at or near the cellular/viral DNA junctions, but significant deletions of the tr sequences have occurred. One of the proviral clones has extensive rearrangements which apparently occurred by nonhomologous recombination. The tail-to-tail arrangement of one of the proviral clones is consistent with limited DNA replication prior to integration by a mechanism similar to that seen in a productive infection. Although there are BamH1 fragments in the cell line we have characterized, which are the right size to have come from a head-to-tail tandem repeat, critical double digests show no evidence for the presence of a head-to-tail tandem repeat of wild-type proviral DNA. Use of probes derived from the flanking cellular DNA enabled us to determine that in this cell line the viral DNA had integrated into a site composed of nonrepetitive sequences.