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Vector development by molecular evolution: a selection procedure for envelope proteins pseudotyping lentiviral particles
RNA VIRUS VECTORS: NEW TECHNOLOGIES major part of the proviral genome has been deleted from these clones.Together, the results broaden our understanding of the precise mechanism of action of CEM15. In conclusion, we believe that these results will be valuable in evaluation of the safety profile of retro- and lentiviral vectors. In addition, these experiments provide sufficient reason to reconsider the necessity of RCR-testing of ex vivo transduced T cells.
332. Dual Gene Transfer and Transgenesis by Novel Lentiviral Vectors Carrying Synthetic BiDirectional Promoters Mario Amendola, Mary Anna Venneri, Elisa Vigna, Luigi Naldini. 1 HSR-TIGET, Milan, Italy; 2HSR-TIGET, Milan, Italy; 3IRCC, Torino, Italy; 4HSR-TIGET, Milan, Italy. Expression of more than one transgene within the same cell allows combining genetic correction with marking, selection, amplification, and conditional elimination of transduced cells. In addition, it is required when gene therapy depends on multiple or synergic genes. Unfortunately, reaching coordinate, high-level dual gene expression in the majority of transduced cells poses significant challenges to current gene transfer technologies. We showed that IRES-based bicistronic lentiviral vectors co-express two transgenes in only a fraction of transduced cells, and that transgene expression was significantly lower when IRES-dependent than when it was capdependent. Thus, selection of transduced cells was required to ensure coordinate expression of both transgenes in all target cells. To overcome these limitations, we developed a novel vector design in which a minimal core promoter was fused upstream and close to an efficient promoter, but in the opposite orientation. The resulting bidirectional promoter mediated coordinate transcription of two divergent mRNA’s. We tested several constructs by changing the trailer core promoter and the driver enhancer/promoter, and found that the bi-directional design significantly enhanced transcription from the trailer core promoter, did not affect downstream expression from the driver promoter, and allowed stable co-expression of two genes from a single vector genome. Strikingly, this new type of vector enabled efficient dual gene transfer and expression in primary hematopoietic progenitors, both of human and mouse origin, tested as immature cells, and after transplantation and differentiation, and in primary T lymphocytes, transduced when proliferating and when non proliferating, after cytokine exposure. Importantly, the new vectors allowed proficient in vivo dual gene transfer and transgenesis. After perivitelline injection of bi-directional vector, we observed robust pan-cellular co-expression of two marker genes in all tissues analyzed of founder and progeny transgenic mice, including brain, liver, kidney, gut, spleen, heart and muscle. Intriguingly, a recent survey of the human genome indicated an abundance of divergently transcribed gene pairs, whose transcription start sites are separated by less than 1 kb. It is likely that many of the promoter elements found between these gene pairs can initiate transcription in both directions, and contain shared elements that regulate both genes. Thus, the synthetic bi-directional promoters that we developed may mimic a well-represented and evolutionary conserved feature of eukaryotic transcription, providing a structural basis for their robust performance. The new lentiviral vectors built around these bi-directional promoters will advance the reach of gene therapy and increase the power of gene-function and target validation studies.
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333. Vector Development by Molecular Evolution: A Selection Procedure for Envelope Proteins Pseudotyping Lentiviral Particles Christoph A. Merten,1 Joern Stitz,1 Klaus Cichutek,1 Christian J. Buchholz.1 1 Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany. Background: Pseudotyping with heterologous envelope proteins is extensively used to generate lentiviral vectors. However, the molecular determinants in the cytoplasmic tails (CTs) of Env proteins governing particle incorporation and membrane fusion are not well characterised. We established a systematic approach allowing the selection of variants mediating optimised transduction rates of lentiviral vectors from libraries of diversified Env proteins. To set up the system we chose the gibbon ape leukaemia virus (GaLV) Env which is unable to mediate transduction of HIV vector particles. However, this block can be overcome by exchanging three amino acids located in the CT of Env. Results: To generate the libraries, bicistronic HIV- or MLVpackagable vectors containing the GaLV env and the neomycin resistance (Neo-R) genes were constructed. The CT was then combinatorially diversified at residues 618, 619, and 623 resulting in libraries comprising up to 106 bacterial clones. For selection, the library was transfected into HIV gag/pol producing cells, and released particles were transferred to fresh cells followed by Neo selection. The GaLV Env vector was then mobilised from Neo resistant cells by gag/pol transfection to initiate further selection cycles. In an alternative approach, we selected for the fusiogenic activity of the Env protein variants. For this, cell-cell fusion was coupled to the release of transducing vector particles having the coding sequences for the particular fusiogenic Env variant encapsidated. Again, Neo selection of cells transduced with the released particles was performed to amplify the coding sequences of the selected variants. Sequence analysis of the env genes present in about thirty Neo resistant clones obtained from both types of selection procedures revealed several distinct CT variants all unrelated to those of GaLV or other C-type retroviruses. Interestingly, all fusiogenic variants had retained expression of the fusion inhibitory R-peptide located at the C-terminus of the CT. The most frequently selected variants showed considerably enhanced incorporation and cell surface expression rates. Moreover, due to the selection procedures the transduction efficiencies as well as the ability to induce syncytia formation were drastically improved as compared to the parental GaLV Env. Conclusions: This is the first proof of principle for the optimisation of lentiviral vectors by evolving optimal Env proteins for a given type of vector particles. Moreover, the system is of high relevance for the improvement of fusiogenic proteins that were recently described as anti-tumoural agents.
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
332. Dual Gene Transfer and Transgenesis by Novel Lentiviral Vectors Carrying Synthetic BiDirectional Promoters Mario Amendola, Mary Anna Venneri, Elisa Vigna, Luigi Naldini. 1 HSR-TIGET, Milan, Italy; 2HSR-TIGET, Milan, Italy; 3IRCC, Torino, Italy; 4HSR-TIGET, Milan, Italy. Expression of more than one transgene within the same cell allows combining genetic correction with marking, selection, amplification, and conditional elimination of transduced cells. In addition, it is required when gene therapy depends on multiple or synergic genes. Unfortunately, reaching coordinate, high-level dual gene expression in the majority of transduced cells poses significant challenges to current gene transfer technologies. We showed that IRES-based bicistronic lentiviral vectors co-express two transgenes in only a fraction of transduced cells, and that transgene expression was significantly lower when IRES-dependent than when it was capdependent. Thus, selection of transduced cells was required to ensure coordinate expression of both transgenes in all target cells. To overcome these limitations, we developed a novel vector design in which a minimal core promoter was fused upstream and close to an efficient promoter, but in the opposite orientation. The resulting bidirectional promoter mediated coordinate transcription of two divergent mRNA’s. We tested several constructs by changing the trailer core promoter and the driver enhancer/promoter, and found that the bi-directional design significantly enhanced transcription from the trailer core promoter, did not affect downstream expression from the driver promoter, and allowed stable co-expression of two genes from a single vector genome. Strikingly, this new type of vector enabled efficient dual gene transfer and expression in primary hematopoietic progenitors, both of human and mouse origin, tested as immature cells, and after transplantation and differentiation, and in primary T lymphocytes, transduced when proliferating and when non proliferating, after cytokine exposure. Importantly, the new vectors allowed proficient in vivo dual gene transfer and transgenesis. After perivitelline injection of bi-directional vector, we observed robust pan-cellular co-expression of two marker genes in all tissues analyzed of founder and progeny transgenic mice, including brain, liver, kidney, gut, spleen, heart and muscle. Intriguingly, a recent survey of the human genome indicated an abundance of divergently transcribed gene pairs, whose transcription start sites are separated by less than 1 kb. It is likely that many of the promoter elements found between these gene pairs can initiate transcription in both directions, and contain shared elements that regulate both genes. Thus, the synthetic bi-directional promoters that we developed may mimic a well-represented and evolutionary conserved feature of eukaryotic transcription, providing a structural basis for their robust performance. The new lentiviral vectors built around these bi-directional promoters will advance the reach of gene therapy and increase the power of gene-function and target validation studies.
S126
333. Vector Development by Molecular Evolution: A Selection Procedure for Envelope Proteins Pseudotyping Lentiviral Particles Christoph A. Merten,1 Joern Stitz,1 Klaus Cichutek,1 Christian J. Buchholz.1 1 Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany. Background: Pseudotyping with heterologous envelope proteins is extensively used to generate lentiviral vectors. However, the molecular determinants in the cytoplasmic tails (CTs) of Env proteins governing particle incorporation and membrane fusion are not well characterised. We established a systematic approach allowing the selection of variants mediating optimised transduction rates of lentiviral vectors from libraries of diversified Env proteins. To set up the system we chose the gibbon ape leukaemia virus (GaLV) Env which is unable to mediate transduction of HIV vector particles. However, this block can be overcome by exchanging three amino acids located in the CT of Env. Results: To generate the libraries, bicistronic HIV- or MLVpackagable vectors containing the GaLV env and the neomycin resistance (Neo-R) genes were constructed. The CT was then combinatorially diversified at residues 618, 619, and 623 resulting in libraries comprising up to 106 bacterial clones. For selection, the library was transfected into HIV gag/pol producing cells, and released particles were transferred to fresh cells followed by Neo selection. The GaLV Env vector was then mobilised from Neo resistant cells by gag/pol transfection to initiate further selection cycles. In an alternative approach, we selected for the fusiogenic activity of the Env protein variants. For this, cell-cell fusion was coupled to the release of transducing vector particles having the coding sequences for the particular fusiogenic Env variant encapsidated. Again, Neo selection of cells transduced with the released particles was performed to amplify the coding sequences of the selected variants. Sequence analysis of the env genes present in about thirty Neo resistant clones obtained from both types of selection procedures revealed several distinct CT variants all unrelated to those of GaLV or other C-type retroviruses. Interestingly, all fusiogenic variants had retained expression of the fusion inhibitory R-peptide located at the C-terminus of the CT. The most frequently selected variants showed considerably enhanced incorporation and cell surface expression rates. Moreover, due to the selection procedures the transduction efficiencies as well as the ability to induce syncytia formation were drastically improved as compared to the parental GaLV Env. Conclusions: This is the first proof of principle for the optimisation of lentiviral vectors by evolving optimal Env proteins for a given type of vector particles. Moreover, the system is of high relevance for the improvement of fusiogenic proteins that were recently described as anti-tumoural agents.
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy