SBIR-STTR Award

The goal of this proposal is to engineer safer more efficacious vectors for use in human gene therapy. The development of safer and more efficient retroviral vectors has significant commercial potential in that these product vectors could become the standard for all retroviral gene therapy trials. Retroviral vectors have been utilized in clinical trials since1990, but poor efficacy remains a major problem. Not only are genes poorly expressed in many cell types, but the vectors are often silenced after a short period of time. Some tissue specificity can be obtained by using tissue-specific promoters, but traditional vectors do not allow genes to be expressed in a regulated manner nor in a development-specific manner. Furthermore, because retroviral vectors insert essentially randomly into transcriptionally active sites, insertional mutagenesis has always been a concern. With the occurrence of a leukemia-like disease in two French SCID-X1 patients who had been successfully treated with a murine retroviral vector, efforts to make retroviral vectors safer has taken on increased priority. We propose the following hypothesis: Retroviral vectors can be made safer and more efficacious by deleting viral cis-regulatory sequences and replacing them with human (or other eukaryotic) regulatory sequences. By removing the enhancer/promoter in the 3' LTR (i.e., using a SIN backbone), inserting a human locus control region together with its endogenous promoter, and providing insulator sequences, the potential for regulated gene expression would be greatly increased while the potential for activation of downstream oncogenes would be greatly reduced. The silencing of transduced genes would be minimized based on the speculation that mammalian cells may have a mechanism for recognizing viral regulatory sequences, and silencing them. Furthermore, the addition of a suicide gene translated from an IRES would add an additional safety feature. We propose to develop the design rules that will allow the construction of this new generation of retroviral and lentiviral vectors. These vectors would increase the efficacy and safety of gene therapy vectors to be used in clinical trials.

Thesaurus Terms:
Lentivirus, Retroviridae, biotechnology, gene induction /repression, gene therapy, genetic manipulation, transfection /expression vector gene expression, genetic promoter element, genetic regulatory element, genetic transduction, therapy adverse effect human genetic material tag, laboratory mouse

Phase I funding for this project supported the successful development of the backbone for a new generation of retroviral gene therapy vectors that contain safety and efficacy features designed to overcome the problems that have been found with retroviral vectors used in current clinical trials. The model disease targeted is the well-studied genetic disease Adenosine Deaminase Deficient Severe Combined Immunodeficiency (ADA-SCID). Phase I work has produced a vector backbone with all viral regulatory sequences involved in gene expression removed and replaced with tissue-specific human (or eukaryotic) regulatory sequences. Specifically the vector has the authentic human ADA Locus Control Region (LCR) and the human ADA promoter in a Self-Inactivating (SIN) backbone, together with additional sequences to improve safety and stability. The vector has a high titer and was shown to express GFP in a tissue-specific (i.e., specific for T cells) manner that suggests that the ADA LCR and ADA promoter are functioning properly. The goal of Phase II is to use the vector backbone developed in Phase I to construct clinically useful, commercially viable gene therapy vectors that can be utilized in clinical trials. The safety and efficacy features being developed can be incorporated into any retroviral vectors and therefore are of broad utility. The hypothesis on which this work is based is the following: Retroviral vectors can be made safer and more efficacious by deleting viral c/s-regulatory sequences and replacing them with human (or other eukaryotic) regulatory sequences. We hypothesize that by removing the enhancer/promoter in the 3' LTR (i.e., using a SIN backbone), inserting a human LCR together with its natural endogenous promoter, and providing insulator sequences, the potential for regulated, tissue-specific, physiological gene expression would be greatly increased while the potential for activation of downstream oncogenes would be greatly decreased. Insertion of a suicide gene translated from an IRES would provide an additional safety feature. Retroviral vectors built using these principles would increase the efficacy and safety of gene therapy vectors to be used in clinical trials. Commercialization Aspects: The achievement of the goals outlined in this Phase II proposal will provide a series of superior retroviral gene therapy vector that will be highly suitable for human gene therapy clinical trials. To commercialize these vectors, Neumedicines plans to partner with companies who are specifically targeting gene-based diseases. Such licensing arrangements will provide for use of these vectors by our partners in a field-exclusive manner, and consequently, will provide a critical step in bringing the technology to its marketable fruition.