In utero delivery of rAAV2/9 induces neuronal expression of the transgene in the brain: towards new models of Parkinson's disease.

Animal models are essential tools for basic pathophysiological research as well as validation of therapeutic strategies for curing human diseases. However, technical difficulties associated with classical transgenesis approaches in rodent species higher than Mus musculus have prevented this long-awaited development. The availability of viral-mediated gene delivery systems in the past few years has stimulated the production of viruses with unique characteristics. For example, the recombinant adeno-associated virus serotype 9 (rAAV2/9) crosses the blood-brain barrier, is capable of transducing developing cells and neurons after intravenous injection and mediates long-term transduction. Whilst post-natal delivery is technically straightforward, in utero delivery bears the potential of achieving gene transduction in neurons at embryonic stages during which the target area is undergoing development. To test this possibility, we injected rAAV2/9 carrying either A53T mutant human α-synuclein or green fluorescent protein, intracerebroventricularly in rats at embryonic day 16.5. We observed neuronal transgene expression in most regions of the brain at 1 and 3 months after birth. This proof-of-concept experiment introduces a new opportunity to model brain diseases in rats.

Adeno-associated virus site-specifically integrates into a muscle-specific DNA region.

The nonpathogenic human virus adeno-associated virus type 2 (AAV) has evolved the potentially unique strategy to establish latency by site-specifically integrating its genome into human chromosome 19 (19q13.3-qter) at a locus designated AAVS1. This nonhomologous, site-specific recombination of viral DNA with the human genome provides a basis for developing targeted gene therapy vectors. To assess whether the region surrounding AAVS1 might have contributed to the selection of the specific integration site, we have investigated this locus. Here, we show that AAVS1 is closely linked to the slow skeletal troponin T gene, TNNT1, which has been mapped previously to 19q13.4. In support of this idea, we demonstrate that site-specific AAV DNA integration can result in the formation of TNNT1-AAV junctions. The question now arises whether muscle represents a natural target tissue for latent AAV infection. This possibility is of additional interest in view of recent observations that muscle tissue is particularly well suited for AAV-mediated gene transfer. The question also occurs whether latent infection by AAV can lead to phenotypic changes of the multinucleated muscle fiber cells.

Presence of integrated DNA sequences of adeno-associated virus type 2 in four cell lines of human embryonic origin.

The human helper virus-dependent parvovirus adeno-associated virus (AAV) has been found in human female genital tissues including material from first trimester miscarriage. In the latter case, AAV type 2 (AAV-2) DNA and viral proteins were detected mainly in the trophoblast cell layer of placenta. In this report, we present evidence that AAV DNA is also present in established human trophoblast cell lines (JEG-3, JAr, BeWo) and in the human amnion cell line FL. In cells of these lines, AAV-2 DNA could be detected both by PCR and Southern blot analysis. Restriction enzyme analysis indicated that AAV DNA was integrated into the host cell genome. Although the cell lines supported AAV replication when infected with AAV-2 and adenovirus type 2 (Ad2) as a helper virus, superinfection with Ad2 alone did not induce replication of AAV DNA, i.e. it failed to rescue AAV from its integrated state. This is probably due to rearrangements within the integrated AAV genome. The presence of AAV DNA in cells derived from human embryonic tissue corroborates the suggestion that human embryonic tissue may be one of the targets of AAV infection.

Human genital tissues containing DNA of adeno-associated virus lack DNA sequences of the helper viruses adenovirus, herpes simplex virus or cytomegalovirus but frequently contain human papillomavirus DNA.

The detection of DNA of the helper virus-dependent adeno-associated virus type 2 (AAV-2) in biopsies of material from spontaneous abortion and in tissue samples from the uterus raises the question of whether sequences of known helper viruses can be detected simultaneously within the same specimen despite the lack of histological evidence for the presence of lytic viruses. Therefore, we performed PCR analyses with primers detecting DNA sequences of viruses (adenovirus, herpes simplex virus and human cytomegalovirus) known for their helper activity in the replication of adeno-associated viruses. In addition, PCR was performed to detect DNA of human papillomaviruses (HPV), which were recently shown to be able to help AAV replication in vitro. In no cases were sequences of the known helper viruses found. However, HPV DNA was detected in approximately 60% of paraffin sections from uterus biopsies and cervical lesions containing AAV DNA and in approximately 70% of material from early miscarriage. This finding suggests that HPV may be a helper virus for AAV.