416. Transient Gene Expression by Non-Integrating Lentiviral (NIL) Vectors

Abstract

The ability to express genes transiently in stem cells would have a wide range of useful applications; for example the introduction of genes that could stimulate cell division, induce commitment to pathways of differentiation, or provide transient resistance to chemotherapeutic agents. Despite the potential usefulness of transient gene expression in hematopoietic stem cells (HSCs), there are still few methods that achieve gene delivery to these cells at high levels. Vectors based on attenuated Retroviruses and Lentiviruses have been used to efficiently and stably introduce genes into cells for long term expression. Their ability to stably transduce cells relies on the fact that their genome encodes an integrase protein that specifically recognizes sequences in the viral long terminal repeats (LTRs) and catalyses their insertion into host chromosomes. Due to their high efficiency of gene delivery and relatively low toxicity, we hypothesize that Non-Integrating Lentiviral (NIL) vectors may also be extremely useful in situations where transient gene expression is required. NIL vectors are lentiviral vectors harboring combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences located in the viral LTRs. Therefore, NIL vectors are designed to disable integration without affecting entry into cells, thus promoting transient gene expression. We have used these NIL vectors to transduce both HSCs and cell lines in order to assess their efficacy, as measured through transient eGFP expression. Our data demonstrate that transductions using the most effective NIL vectors harboring both LTR and integrase mutations resulted in initial high level transient eGFP expression (~90%), diminishing towards background in <1 month. This is in contrast to the wild-type vector which causes continued expression of eGFP (up to 3 months in cell lines). Encouragingly, NIL vectors performed equally well in primary human CD34+ cells as they did in cell lines. Southern blot analyses of transduced Jurkat cells depicted the loss of detectable NIL vector sequence after 1 month. Additionally the Southern analyses revealed important information regarding the frequency of formation of 1 or 2 LTR circles in comparison to linear DNA, which are typical molecular species formed by such non-integrating viral vectors. Through PCR and sequencing we have studied the number and nature of integrations of the NIL vectors in pools and colonies of Jurkat cells and compared this information to results from Jurkat cells exposed to the wild-type lentiviral vector. We believe that these studies may lead to the development of a vector system capable of highly efficient, but transient gene expression in human stem and mature cells.