No abstract

No abstract

Adenovirus type 5 deletion mutants that lack portions of their cis-acting DNA encapsidation signal synthesized nearly normal levels of viral DNA and late polypeptides but failed to efficiently package the DNA into virus particles. A series of mutant viruses carrying small deletions were produced and used to identify a repeated element (AGTAAATTTGGGC and AGTAAGATTTGGCC) as a key component of the packaging signal. One copy of the repeat was sufficient to signal efficient packaging. The packaging domain could function near either end of the viral chromosome but was no longer active when moved several hundred base pairs toward the interior of the DNA molecule.

Effective clinical delivery of gene therapy to the heart requires understanding and design of complex biological systems to deliver therapeutic gene expression. The development of vectors that specifically target the myocardium, in particular bioengineered recombinant viruses, has improved the efficiency of gene delivery to the heart. These tools, coupled with advances in selection and design of the genetic payload, have led to effective cardiac gene therapy in preclinical models. This technology is currently translating to the clinic with a new wave of gene therapy trials for myocardial disease.

Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.