"We hope our vector will be curative, (while) requiring less integration per genome than current vectors," Drs. Laura Breda and Stefano Rivella of Children's Hospital of Philadelphia told Reuters Health by email "If this is the case, we would like to evaluate novel approaches using reduced conditioning regimens and, eventually, direct delivery of the vector to the bone marrow of patients, bypassing myeloablation."
Currently, gene therapy trials for beta-globinopathies involve transferring 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," the researchers write in Molecular Therapy.
Instead, the team generated five novel lentiviral vectors and, for comparative purposes, three vectors that were similar to those already used in clinical trials.
In a series of in vitro and in vivo screening experiments, they discovered that one of their vectors, ALS20, expressed significantly higher transgenic hemoglobin levels than the three currently used to treat beta-globinopathies.
Specifically, compared to the current vectors, ALS20 produced 157%, 84%, and 40% more adult hemoglobin. The findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients.
Further, bone marrow transplantation studies in beta-thalassemia mouse models showed that ALS20 was curative at less than one copy per genome. And, in xenograft experiments, injecting human CD34+ cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment.
Summing up, the authors state, "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 reducing the intensity of myeloablation."
Dr. James Taylor, Director of the Center for Sickle Cell Disease at Howard University in Washington, DC, commented in an email to Reuters Health, "There are two concerns: first, can the vector delivering the new gene make enough of itself to correct the disease process? The second is long-term safety."
Previous work has relied upon about four copies of the lentivirus being integrated into each cell, he said, and "each integration event has a risk for producing new mutations that could result terrible outcomes like cancer."
By contrast, ALS20 "turns on the viral gene much more efficiently in red cells - and in principle could rely upon <1 integrated viral transgene per cell genome," he said. "So, this approach in theory could more effectively correct the disease process AND at the same time reduce the risk of devastating long-term complications."
Further, he noted, "75% of those with sickle cell disease are in resource-poor countries in Africa. If we can design a therapy that requires less intense chemotherapy and (provides) greater safety, then it might be possible to use such a strategy in Africa. The current vectors and requirements for intense chemotherapy are not practical where sickle cell is most common."
While ALS20 is promising, he added, "these studies were done in a beta thalassemia model, so it is unclear if the same results would be seen in a sickle cell disease model. We will also not know the long-term safety or if patient outcomes are better than standard treatments for either beta thalassemia or sickle cell disease for a number of years."
Dr. Srila Gopal, Clinical Assistant Professor, Division of Hematology/ Oncology at the University of California San Diego, also commented by email to Reuters Health. "There are several lentiviral-based vectors, and more recently, a CRISPR- Cas9-based gene editing strategy (https://bit.ly/3tKRgRw) that obviates the need for a viral vector, which are being developed for achieving 'cure' in hemoglobinopathies. How this compares to the traditional lentiviral based vectors remains to be seen."
Like Dr. Walker, she noted safety concerns, including "the theoretical risk for viral genome integration into the host DNA, thereby causing insertional oncogenesis, and the potential long-term effects from high-dose chemotherapy given as part of conditioning regimens prior to gene transfer."
"Gene therapy in hemoglobinopathies offers hope for cure, but requires careful patient selection," she added. "The financial burden of this treatment remains unknown, but it likely to not be trivial."
The researchers are finalizing an IND application and hope to treat the first patients by the end of 2021 or beginning of 2022.
SOURCE: https://bit.ly/3rKxDHd Molecular Therapy, online January 29, 2021
By Marilynn Larkin
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