Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression

Abstract

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. transActing AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.

Direct Gene Transfer Into Human Epileptogenic Hippocampal Tissue with an Adeno-Associated Virus Vector: Implications for a Gene Therapy Approach to Epilepsy

“…One of the most promising of these approaches is the application of DNA-containing viral vectors that permit direct introduction of a cloned gene into neurons andor glia, resulting in its expression into a functional protein. Three broad categories of DNA viruses are currently being used for CNS gene delivery; these are based on HSV-1, Adenovirus and, most recently, AAV (1-As a gene therapy vector, AAV has several desirable features (2, 27,36). Notably, unlike wild-type HSV-1 and Adenovirus, which can cause disseminated infections in humans, AAV has not been associated with any human disease, although more than three quarters of the United States population is seropositive for the AAV virus (36,39).…”

“…Three broad categories of DNA viruses are currently being used for CNS gene delivery; these are based on HSV-1, Adenovirus and, most recently, AAV (1-As a gene therapy vector, AAV has several desirable features (2, 27,36). Notably, unlike wild-type HSV-1 and Adenovirus, which can cause disseminated infections in humans, AAV has not been associated with any human disease, although more than three quarters of the United States population is seropositive for the AAV virus (36,39). In addition, AAV is a defective virus, unable to replicate in the absence of a helper virus, which commonly is Adenovirus.…”

“…AAV vectors can direct expression of genes in both mitotic and postmitotic, terminally differentiated cells, including neurons, whereas Adenovirus preferentially transfects dividing cells (14,26,29). A further ad-5, [13][14][15][22][23][24][25][26][27]36,39). vantage of AAV is that pure, helper-free stocks can readily be made with a novel packaging system that completely eliminates production of immunogenic viral proteins, a feature not currently available with either HSV-1 or Adenovirus (36).…”

“…The construction of AAVlacZ has been described previously (27,36). The AAV plasmid encoding the lacZ gene was developed by subcloning the cytomegalovirus (CMV) immediate-early promoter, lacZ, and a simian virus 40 (SV40) polyadenylation signal between the terminal repeats of the AAV genome in plasmid psub201.…”

“…We have therefore initiated a series of studies examining the potential of an alternative viral vector system based on AAV to introduce and cause the expression of genes in the CNS. AAV has several desirable features: It is a DNA virus, not directly associated with any human disease; it can direct transgene expression in numerous cell types, including postmitotic neurons; and it may stably integrate into the host chromosome (2, 27,[36][37][38][39]. Furthermore, helper virus-free vector stocks that do not express any viral proteins, rendering an immune response less likely, can be obtained.…”