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Cas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease.

ABSTRACT: Gene editing with the CRISPR-Cas9 system could revolutionize hematopoietic stem cell (HSC)-targeted gene therapy for hereditary diseases, including sickle cell disease (SCD). Conventional delivery of editing tools by electroporation limits HSC fitness due to its toxicity; therefore, efficient and non-toxic delivery remains crucial. Integrating lentiviral vectors are established for therapeutic gene delivery to engraftable HSCs in gene therapy trials; however, their sustained expression and size limitation preclude their use for CRISPR-Cas9 delivery. Here, we developed a Cas9 protein delivery non-integrating lentiviral system encoding guide RNA and donor DNA, allowing for transient endonuclease function and inclusion of all editing tools in a single vector (all-in-one). We demonstrated efficient one-time correction of the SCD mutation in the endogenous βs-globin gene up to 42% at the protein level (p < 0.01) with the Cas9 protein delivery non-integrating lentiviral all-in-one system without electroporation. Our findings improve prospects for efficient and safe genome editing.

SUBMITTER: Uchida N

PROVIDER: S-EPMC8005818 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The efficacy and biosafety of lentiviral gene transfer is influenced by the design of the vector. To this end, properties of lentiviral vectors can be modified by using cis-acting elements such as the modification of the U3 region of the LTR, the incorporation of the central flap (cPPT-CTS) element, or post-transcriptional regulatory elements such as the woodchuck post-transcriptional regulatory element (WPRE). Recently, several studies evaluated the influence of the incorporation of insulators into the integrating lentiviral vector genome on transgene expression level and position effects. METHODS:In the present study, the influence of the matrix attachment region (MAR) of the mouse immunoglobulin-? (Ig-?) or the chicken lysozyme (ChL) gene was studied on three types of HIV-1-derived lentiviral vectors: self-inactivating (SIN) lentiviral vectors (LV), double-copy lentiviral vectors (DC) and non-integrating lentiviral vectors (NILVs) in different cell types: HeLa, HEK293T, NIH-3T3, Raji, and T Jurkat cell lines and primary neural progenitors. RESULTS AND DISCUSSION:Our results demonstrate that the Ig-? MAR in the context of LV slightly increases transduction efficiency only in Hela, NIH-3T3 and Jurkat cells. In the context of double-copy lentiviral vectors, the Ig-? MAR has no effect or even negatively influences transduction efficiency. In the same way, in the context of non-integrating lentiviral vectors, the Ig-? MAR has no effect or even negatively influences transduction efficiency, except in differentiated primary neural progenitor cells.The ChL MAR in the context of integrating and non-integrating lentiviral vectors shows no effect or a decrease of transgene expression in all tested conditions. CONCLUSIONS:This study demonstrates that MAR sequences not necessarily increase transgene expression and that the effect of these sequences is probably context dependent and/or vector dependent. Thus, this study highlights the importance to consider a MAR sequence in a given context. Moreover, other recent reports pointed out the potential effects of random integration of insulators on the expression level of endogenous genes. Taken together, these results show that the use of an insulator in a vector for gene therapy must be well assessed in the particular therapeutic context that it will be used for, and must be balanced with its potential genotoxic effects.

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Cas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease.

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