Infection Efficiency of Human and Mouse Embryonic Stem Cells Using Adenoviral and Adeno-Associated Viral Vectors

Abstract

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors

“…Compared with adeno-associated virus vectors, HDAdVs are more efficient in gene delivery into hES cells (20), show a higher ratio of targeted to random integration, and accommodate larger and more complex targeting cassettes.…”

“…Although an E1-deleted AdV was used for gene transfer into hES cells, the transduction efficiency (11%) was low (20). Helper-dependent AdVs (HDAdVs) were originally developed to overcome host immune responses against E1-deleted AdVs in vivo (21,22).…”

Recombinant AAV2 transduction of primitive human hematopoietic stem cells capable of serial engraftment in immune-deficient mice

“…Song et al (29) showed repopulation of hepatectomized murine livers with rAAVtransduced oval cells (29), which possess progenitor activity and display CD34 and CD45 surface markers similar to HSC. rAAV transduction of marrow-derived mesenchymal stem cells (30,31), alveolar stem cells (32), neural progenitor cells (33), and embryonic stem cells (34) has been demonstrated. If primitive quiescent HSCs were more efficiently transduced than dividing progenitors, higher marking frequencies in secondary recipients would be expected, as observed in this study.…”

Library selection and directed evolution approaches to engineering targeted viral vectors

“…Despite these advantageous properties, AAV suffers from several shortcomings, including the inability to target delivery to specific cell types (Muzyczka and Warrington, 2005), inefficient gene delivery to a number of ''nonpermissive'' cell types (Hughes et al, 2002;Ponnazhagan et al, 1996;Smith-Arica et al, 2003;Stacchini et al, 1999), a limited packaging insert size (Dong et al, 1996), and the prevalence of pre-existing immunity to human AAV serotypes in the human population (Moskalenko et al, 2000;Sun et al, 2003).…”