CRISPR/Cas9 Gene Editing in Cell and Gene Therapies

CRISPR/Cas—a system that was initially discovered as a bacterial adaptive immune system used for destroying viral invaders has grown leaps and bounds in the last decade into one of the most revolutionary technologies of modern medicine to have the capability to change an organism’s genome. Although older gene-editing tools are still relevant, such as TALENS and ZFNs, the accuracy, efficiency, performance, and affordability of CRISPR/Cas have made it a popular platform for gene therapies.

CRISPR/Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (the most commonly used Cas protein in gene editing)—offers endless possibilities for programmable genome manipulation ranging from addition and deletion of the gene(s) to specific base-pair modifications. CRISPR technology recently showed promise in preclinical data for the treatment of Duchenne muscular dystrophy in a canine model, in which CRISPR-corrected dystrophin gene seemed to have restored limb function in affected King Charles spaniels.

Dr. Jennifer Doudna, recipient of this year’s Nobel prize in chemistry alongside Dr. Emanuelle Charpentier, noted that her work on CRISPR started as a curiosity-driven research. Doudna and Charpentier found that bacteria used a very precise system for chopping viral DNA, a system that used a couple of RNA molecules to guide a nuclease protein to the target DNA sites. In their seminal work, published in Science, Cas9 nuclease was shown to be guided by dual RNAs—crRNA and tracrRNA—to a DNA sequence complementary to crRNA to introduce double-stranded (ds) cuts.

The ds breaks are subsequently repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). However, the seemingly simple mechanism was about to turn into what may be a unique clinically beneficial technology when an engineered single RNA chimera (instead of the dual RNA system) could guide the Cas9 to make cuts in DNA. The ability to use a single guide RNA (sgRNA) to make site-specific cuts in DNA soon became a breakthrough technology that could be exploited for genome editing.

Today, scientists can use CRISPR/Cas9 to treat genetic diseases beyond the reach of traditional drug-based therapies; in fact, several clinical trials are underway for hematologic cancers and monogenic blood disorders like Beta-Thalassemia, Thalassemia, and sickle cell anemia.

CRISPR/Cas9 Edited T Cell Therapy

Revolutionizing the field of cancer immunotherapy, CRISPR/Cas9, yet again proved to be a powerful platform to optimize engineered T cells’ activity. Scientists combined the DNA editing power of CRISPR/Cas9 with T cells to launch the fourth generation of Chimeric antigen receptor (CAR) T cells to treat various hematologic and solid cancers. CARs are engineered receptors on T cells that recognize specific antigen-expressing cancer cells in an MHC independent manner; however, several limitations, such as insufficient quantity, low quality of autologous T cells, CAR T cell exhaustion, and tumor-suppressive microenvironments plague the efficiency of the engineered T cells. The first CRISPR Phase 1 clinical trial in the US opened in 2018, in which CRISPR/Cas9 was used to edit autologous T cells for immunotherapy against several cancers with relapsed tumors that had exhausted all curative options. These include multiple myeloma, melanoma, synovial sarcoma, and myxoid/round cell liposarcoma.

CRISPR engineered T cells can have a variety of features to make them more suitable therapeutically; such features include

1. Knocking-out of native TCR (T cell receptor) and MHC genes from T cells to develop ‘off-the-shelf’ universal T cells,
2. Addition of transgenic TCR that are targeted to tumor antigens,
3. Elimination of immunosuppressive receptors (such as PD-1 and TGF beta receptor) to increase potency, and
4. Deletions of target genes to avoid the self-killing of CAR T cells.

In fact, the development of universal ‘off-the-shelf’ CAR T cells could be one of the greatest applications of CRISPR in onco-immunotherapy that can potentially eliminate the constraints of patient-derived T cells, which are often difficult to obtain and take relatively longer time to be manufactured into CAR T cells before being used in treatments.

CRISPR/Cas9 for Monogenic Blood Disorders

Monogenic blood disorders caused by small insertions, single base-pair mutations, or deletions in a single gene are good candidates for gene therapies. CRISPR-based gene edits are introduced in the hematopoietic stem cells and are infused back in the patient, correcting the disorder. Sickle cell disease, which is caused by a single base-pair mutation (Glu>Val) in the b-globin subunit of hemoglobin, results in a defective hemoglobin S; β-thalassemia, on the other hand, is caused by different types of mutations in the β-globin gene which causes a loss or reduction in the protein. An increased amount of fetal hemoglobin (HbF)—γ-globin—is the universal strategy to circumvent the problem with low β-globin levels.

Recently, CRISPR Therapeutics in 2018 and Allife Medical Science and Technology Co., Ltd in 2019 have started three clinical trials using CRISPR/Cas9 edited human hematopoietic cells (CTX001) to treat both sickle cell disease and β-thalassemia. In the CRISPR-corrected cells, HbF is increased by reducing BCL11A, which suppresses the γ-globin gene. Specifically, CRISPR/Cas cleaves the erythroid enhancer of the BCL11A gene. One SCD patient was reported to have 46.6% HbF and 95% RBCs expressing HbF after 4 months of CTX001 transfusions, and one β-thalassemia patient is expressing 10.1 g/dL HbF out of 11.9 g/dL total hemoglobin, and almost 99% RBCs expressing HbF after 9 months of the therapy.

CRISPR/Cas9 Editing in vivo

While CRISPR/Cas manipulation of genes is permissible and carefully regulated in somatic cells of the body, germline editing for treatment purposes is controversial and remains illegal in many countries. Germline manipulation of genes not only can pass down through generations but also takes away the decision-making process of the later-born individual. However, the team led by He Jiankui from the University of Science and Technology in Shenzhen, China, has already attempted to CRISPR edit the CCR5 gene in human embryos to confer HIV resistance (HIV uses CCR5 to enter T cells). Besides, there could several undesired mutations in the genome.

A naturally occurring 32-base deletion in the CCR5 gene is known to confer HIV resistance; however, He, with the intent to induce a similar omission in the embryonic DNA, ended up with just double-stranded breaks in the DNA, allowing NHEJ to take over and randomly repair the edits. It remains unknown if the unintentional edits in the CCR5 gene would achieve the natural 32-base deleted version’s effects. Germline and embryonic editing raise many ethical concerns, including if genome editing would be used to enhance typical human traits, such as appearance or intelligence.

Gene therapy began as a feasible and promising treatment modality for genetic diseases that could be cured by replacing the defective gene with a healthy version, delivered through viral vectors. However, adverse effects seen in many treated patients dampened the initial excitement until the real-world applications of CRISPR/Cas9 renewed the lost interest of our scientific community towards gene therapy. With this new technology, it is possible to precisely edit a non-functional gene instead of replacing it entirely, as was the case in traditional gene therapy. However, the limitless opportunities of the CRISPR/Cas system warrants greater caution and deliberation in moving forward as failure to learn from the old lessons may make it harder for the technology to progress into a clinically beneficial treatment modality.

By Sangeeta Chakraborty, Ph.D.

Related Article: The Future of CRISPR: Three Areas The Nobel Prize Winning Tech Will Be Most Impactful

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Sangeeta Chakraborty