Dataset Information

Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

ABSTRACT: Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34⁺ cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.

SUBMITTER: Breda L

PROVIDER: S-EPMC8058492 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

Breda Laura L Ghiaccio Valentina V Tanaka Naoto N Jarocha Danuta D Ikawa Yasuhiro Y Abdulmalik Osheiza O Dong Alisa A Casu Carla C Raabe Tobias D TD Shan Xiaochuan X Danet-Desnoyers Gwenn A GA Doto Aoife M AM Everett John J Bushman Frederic D FD Radaelli Enrico E Assenmacher Charles A CA Tarrant James C JC Hoepp Natalie N Kurita Ryo R Nakamura Yukio Y Guzikowski Virginia V Smith-Whitley Kim K Kwiatkowski Janet L JL Rivella Stefano S

Molecular therapy : the journal of the American Society of Gene Therapy 20210129 4

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intro ...[more]

PMID: 33515514

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Project description:BackgroundHIV-responsive expression vectors are all based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the use of LTR-based reporter vectors to cloned cells, where aberrantly high expressing (HIV-negative) cells can be eliminated. Enhancements in specificity would increase opportunities for expression vector use in detection of HIV as well as in experimental gene expression in HIV-infected cells.ResultsWe have constructed an expression vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites that are efficiently used in human cells. It also contains a reading frame that is removed by cellular splicing activity in the absence of HIV Rev. The vector was incorporated into a lentiviral reporter virus, permitting detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of green fluorescence protein (GFP) reporter and by PCR of reporter transcript following HIV infection. The vector displayed full HIV dependency.ConclusionAs with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel alternative approach to existing methods, in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.

Dataset Information

Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

Publications

Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

Similar Datasets

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