Gene Editing

Virus-specific T cells (VSTs) are highly effective in treating infections after hematopoietic stem cell transplant (HSCT). Graft-versus-host disease is a common complication of HSCT, so many patients receive prophylactic glucocorticoids to suppress immune response and better enable HSC engraftment. Treatment with glucocorticoids can often lead to viral reactivation, which can be life threatening for immunosuppressed patients. Unfortunately, this treatment with glucocorticoids also limits the effect of follow-on VST therapy, as they are lymphocytotoxic and induce apoptosis in T cells. 

In this paper, researchers at MD Anderson Cancer Center describe a GMP-compliant method to manufacture CRISPR gene-edited VSTs that are unaffected by glucocorticoid treatment. These CRISPR-edited VSTs have comparable potency to non-gene edited VSTs but could be co-administered to patients with steroids. The GMP manufacturing workflow applies Aldevron SpyFi™ Cas9 Nuclease as the gene editing reagent due to its high on-target editing efficiency and greatly reduced off-target editing in comparison to wild-type SpCas9. 

https://doi.org/10.1182/bloodadvances.2020001977

Basar, R., Daher, M., Uprety, N., Gokdemir, E., Alsuliman, A., Ensley, E., Ozcan, G., Mendt, M., Hernandez Sanabria, M., Kerbauy, L. N., Nunez Cortes, A. K., Li, L., Banerjee, P. P., Muniz-Feliciano, L., Acharya, S., Fowlkes, N. W., Lu, J., Li, S., Mielke, S., Kaplan, M., … Rezvani, K. (2020). Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells. Blood advances, 4(14), 3357–3367.  

X-linked hyper-IgM Syndrome (HIGM1) is a recessive genetic disorder that causes B cells to overproduce IgM antibodies and underproduce IgG, IgA, and IgE. In a healthy immune response, T cells signal B cells to undergo a process called “class switching recombination” in which the cell produces different classes of antibodies that will bind to a given antigen. HIGM1 is caused by inactivating mutations to the CD40LG gene resulting in disruption of the CD40 cell signaling pathway. Patients suffering from HIGM1 are susceptible to a variety of bacterial infections and have poor prospects for long term survival. 

Correction of relevant mutations in T cells has long been explored as a strategy to cure HIGM1. In this paper, researchers at Vita-Salute San Raffaele University and San Raffaele Telethon Institute for Gene Therapy (SR-TIGET) compared two different gene correction strategies – autologous T cell therapy and hematopoietic stem/progenitor cell (HSPC) therapy. When they detected off-target editing at least one locus, the research team switched to Aldevron SpyFi™ Cas9 Nuclease to reduce this off-target activity below detectable levels. Both T-cell and HPSC therapeutic approaches were effective at rescuing immune response in mouse models, but the research team ultimately concluded that T-cell therapy would be a more promising clinical strategy. 

https://doi.org/10.15252/emmm.202013545

Vavassori, V., Mercuri, E., Marcovecchio, G. E., Castiello, M. C., Schiroli, G., Albano, L., Margulies, C., Buquicchio, F., Fontana, E., Beretta, S., Merelli, I., Cappelleri, A., Rancoita, P. M., Lougaris, V., Plebani, A., Kanariou, M., Lankester, A., Ferrua, F., Scanziani, E., Cotta-Ramusino, C., … Genovese, P. (2021). Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome. EMBO molecular medicine, 13(3), e13545. https://doi.org/10.15252/emmm.202013545 

Jacków, J., Guo, Z., Hansen, C., Abaci, H. E., Doucet, Y. S., Shin, J. U., Hayashi, R., DeLorenzo, D., Kabata, Y., Shinkuma, S., Salas-Alanis, J. C., & Christiano, A. M. (2019). CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells. Proceedings of the National Academy of Sciences of the United States of America, 116(52), 26846–26852. Advance online publication. https://doi.org/10.1073/pnas.1907081116

Induced pluripotent stem cells (iPSCs) show great promise as a therapeutic tool for a wide variety of human diseases. Pluripotent stem cells can differentiate into any human cell type, allowing researchers to create patient-specific cell lines that do not carry a risk of rejection in vivo. iPSCs are derived from adult cells, so their study is less controversial than embryonic stem cells for equivalent conditions. Stem cell therapy shows great promise in treating dystrophic epidermolysis bullosa (EB), a rare recessive genetic disorder caused by mutations in the COL7A1 gene that results in fragile skin and painful skin blistering.

In this paper, researchers at Columbia University use CRISPR/Cas9 technology to correct COL7A1 mutations in patient iPSCs. Gene-modified cells were differentiated into keratinocytes and fibroblasts before transplantation and engraftment into a mouse model. Transplanted cells successfully rescued the wild-type skin phenotype and did not show evidence of tumorigenesis, a documented concern for iPSC therapeutics. The research team applied Aldevron SpyFi™ Cas9 Nuclease due to the reduction in off-target editing and equivalent on-target performance in comparison to wild-type SpCas9.