Non-integrating Lentiviral Vectors Research Articles

Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo

Lentivirus-derived vectors are among the most promising viral vectors for gene therapy currently available, but their use in clinical practice is limited by the associated risk of insertional mutagenesis. We have overcome this problem by developing a nonintegrative lentiviral vector derived from HIV type 1 with a class 1 integrase (IN) mutation (replacement of the 262RRK motif by AAH). We generated and characterized HIV type 1 vectors carrying this deficient enzyme and expressing the GFP or neomycin phosphotransferase transgene (NEO) under control of the immediate early promoter of human CMV. These mutant vectors efficiently transduced dividing cell lines and nondividing neural primary cultures in vitro. After transduction, transient GFP fluorescence was observed in dividing cells, whereas long-term GFP fluorescence was observed in nondividing cells, consistent with the viral genome remaining episomal. Moreover, G418 selection of cells transduced with vectors expressing the NEO gene showed that residual integration activity was lower than that of the intact IN by a factor of 500-1,250. These nonintegrative vectors were also efficient in vivo, allowing GFP expression in mouse brain cells after the stereotactic injection of IN-deficient vector particles. Thus, we have developed a generation of lentiviral vectors with a nonintegrative phenotype of great potential value for secure viral gene transfer in clinical applications.

Transient Gene Expression by Nonintegrating Lentiviral Vectors

Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34+ hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (∼90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 104-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.

Effective gene therapy with nonintegrating lentiviral vectors

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.

458. Development of Non-Integrating and Site- Specifically Integrating Lentiviral Vectors

The lentiviral genome non-specifically integrates into the human genome and this can lead to insertional mutagenesis and potentially cell transformation. We are using two strategies to eliminate the risk of insertional mutagenesis thereby improving the safety and efficacy of lentiviral gene therapy vectors. The first is to develop non-integrating lentiviral vectors and the second is to target integration to a non-pathogenic site in the human genome.

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

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.