REVOLUTIONARY GENE THERAPY: CRISPR-Cas9 AND rAAV6 FOR TREATING SEVERE COMBINED IMMUNODEFICIENCY (SCID)

 

Severe combined immunodeficiency (SCID) is a group of very rare monogenic disorders that is caused by defects in both cellular and humoral adaptive immunity. In this case, patients are born healthy but due to the body being susceptible to pathogens, consequent infection in the body occurs which untreated can be fatal. However, scientists have created a gene-editing strategy using CRISPR-Cas9 and recombinant adeno-associated virus serotype 6 (rAAV6) to correct the RAG2 gene in CD34+ hematopoietic stem and progenitor cells (HSPCs). This approach aims to treat patients with RAG2-Severe Combined Immunodeficiency (SCID).

The Recombination-activating genes 1 and 2 (RAG1 and RAG2) are two genes located close to each other on chromosome 11 by a distance of approximately 12 kb. These genes are essential for the development of diversity in B and T cells. The RAG1 and RAG2 proteins catalyze the DNA double-strand breaks at specific sites called recombinant signalling sequences (RSSs). RSSs are located at the junction between variable (V), diversity (D), and joining (J) gene segments. These gene segments are necessary for the receptors to recognize and bind to specific antigens, which are foreign substances or pathogens. When the RAG proteins create these double-strand breaks, they initiate the process of genetic recombination. Once the double-strand breaks are created by RAG1 and RAG2, the cellular machinery repairs these breaks by joining the gene segments together in a unique and random manner. This random joining of gene segments leads to the generation of a highly diverse set of TCR and BCR receptors known as V(D)J Recombination. This process generates diversity in the receptors used by these cells, namely the T-cell receptor (TCR) for T cells and the B-cell receptor (BCR) for B cells. The diversity in the TCR and BCR receptors has a crucial role in allowing the immune system to recognize and respond to various pathogens and foreign substances effectively. 

So RAG1 and RAG2 genes are the key players in the development of B and T cells which initiate the V(D)J recombination which eventually generates the diverse repertoire of T-cell receptors (TCRs) and B-cell receptors (BCRs). However, overexpression or misregulation of the RAG genes can lead to genomic instability. This can result in the formation of translocations and deletions in cancer-causing genes, potentially leading to lymphocyte malignancies. Unsuccessful termination of RAG1 and RAG2 expression leads to atypical thymus development, aberrant lymphatic system, and immunodeficiency. Patients with variations in RAG genes may have a complete absence or significant reduction of T and B cells known as the T-B-NK+ immune phenotype. 

At present, the only solution for SCID patients is an allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-matched donor. The alternative treatment is haploidentical HSCT which reduces the survival rate to less than 80%. Although finding an HLA-matched donor is rare, after a successful HSCT, there is a high risk of graft-versus-host disease.

One of the promising alternatives involves genetically modifying the patient's own CD34+ hematopoietic stem and progenitor cells (HSPCs) in a laboratory setting (ex-vivo) and then reintroducing them into the patient's body for an autologous hematopoietic stem cell transplant (HSCT). CD34+ HSPCs are particularly well-suited for gene therapy applications due to their remarkable ability to regenerate the immune system even when introduced in small numbers. In the past, researchers have explored using viral vectors like lentiviral (LV) or gammaretroviral (γRV) vectors to deliver therapeutic genes into the patient's CD34+ HSPCs for ex-vivo editing. However, there have been safety concerns associated with the use of γRV vectors, particularly in cases where they led to the activation of proto-oncogenes, resulting in some patients developing leukaemia. 

In light of these concerns, the delivery of therapeutic genes via a targeted HDR-mediated genome-editing approach, which combines Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9), along with recombinant adeno-associated virus serotype 6 (rAAV6), is considered a promising option for addressing RAG-SCIDs by the scientists. While rAAV6 offers several advantages over lentiviral (LV) and gammaretroviral (γRV) vectors, it has been previously demonstrated that rAAV6 vectors can trigger a DNA damage response (DDR), and the extent of this response is directly related to the amount of virus used, known as the multiplicity of infection (MOI). To ensure the therapeutic relevance of a CRISPR-Cas9/rAAV6 genome-editing strategy, a delicate balance must be maintained. This balance is aimed at achieving high-quality HDR while minimizing the viral load as much as possible. Consequently, various donor constructs were investigated to determine the optimal rAAV6 donor design for efficient HDR.

In this study, a CRISPR-Cas9/rAAV6 genome-editing strategy was described, involving the replacement of the entire coding sequence (CDS) of RAG2 with a corrective transgene in CD34+ HSPCs. The goal was to maintain the native regulatory elements within the RAG2 locus, which encompassed the upstream promoter regions, an intergenic silencer sequence between RAG1 and RAG2, and evolutionarily conserved genes located within the intronic sequences of the RAG1/2 locus, believed to contribute to gene regulation.

To closely mimic the endogenous expression of the RAG2 gene, three correction donors were designed. One donor retained the 5' promoter and regulatory elements, along with the 3' untranslated region (UTR) and downstream region. The other two donors were engineered to enhance transgene expression by preserving the native 5' UTR while introducing synthetic 3' UTRs in the form of WPRE-BGHpA and BGHpA sequences.

Developing a proof-of-concept gene correction therapy often requires a substantial amount of patient samples, but due to the limited survival of untreated SCID patients beyond infancy, obtaining these samples is challenging. To address this, the researchers used fluorescence-activated cell sorting (FACS) to enrich engineered genotypes in CD34+ hematopoietic stem and progenitor cells (HSPCs) from healthy donors, simulating single-allelic gene-correction therapies for RAG2-SCID. By introducing a diverged codon-optimized RAG2 (dcoRAG2) cDNA cassette in one allele and knocking out the second allele with a cassette disrupting the green fluorescent protein (GFP) gene, the researchers mimicked monoallelic correction in SCID-patient cells. This study demonstrates the simulation of RAG2 gene correction in CD34+ HSPCs from healthy donors, achieving it by completely replacing the native coding sequence.

By maintaining the native regulatory components and intronic sequences, their method accurately replicates the natural gene expression levels, consequently mitigating the potential dangers associated with uncontrolled gene expression. This innovative approach, characterized by the replacement of entire coding sequences or exons while safeguarding essential regulatory elements, offers optimism for individuals afflicted with RAG2-SCID and shows potential as a treatment strategy for a range of other genetic conditions.

REFERENCE

Allen D, Knop O, Itkowitz B, Kalter N, Rosenberg M, Iancu O, et al. CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications. Nature Communications. 2023;14(1). doi:10.1038/s41467-023-42036-5