One of the effective ways of treating autoimmune disease is to identify the “signature” of offending genes (their “gene expression” or the number of RNA molecules they are producing), which is abnormal in autoimmune (and cancer) genes. This identification is accomplished using a technique called “single-cell RNA sequencing” (scRNA-seq), or more specifically, TIDE (for Tumor Immune Dysfunction and Exclusion) for autoimmune genes. With this information, a revolutionary procedure called CRISPR-Cas9 (“Clustered regularly interspaced short palindromic repeats” – a family of DNA sequences found in the genomes of prokaryotic organisms, i.e., organisms where the DNA is in the cell cytoplasm rather than its nucleus – this is explained in a bit more understandable language ahead, so feel free to forget this last sentence) and Cas9, an enzyme sometimes referred to as “the scissor protein.” In essence, the procedure is an RNA-guided genome editing technology being used to reengineer T cells.
Similar to the way bacteria defend against viral invasion, CRISPR-Cas9 is used to induce genome edits by creating targeted DNA breaks that will trigger specific DNA repair. When considering “next-generation” genetic processing (“central dogma of molecular biology” – see Blog #10), it can also control the transcriptional output of genes or alter genome sequences using a process of nucleotide base editing. As these technologies continue to mature, it is becoming increasingly possible to efficiently and accurately alter cellular genomes.
The CRISPR-Cas9 system (Figure 5.5) creates a small piece of RNA (Cas9) with a short “guide” sequence that attaches (binds) to a specific target sequence of DNA identified by NGS (next generation sequencing – see Blog #10) in a genome. The RNA also binds to the Cas9 enzyme and is used to recognize the DNA sequence. The Cas9 enzyme, acting as a “scissor,” cuts the DNA at the targeted location. Once the DNA is cut, the cell’s DNA uses its repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence. It was first thought that the stitching back together of the genetic material after the CRISPR-Cas9 procedure was random. But subsequent studies using a trained machine learning (ML) algorithm called inDelphi to predict repairs made to DNA snipped with Cas9 confirmed that the edits aren’t random at all.
It is worth noting here that in October 2020, the Nobel Prize in Chemistry was awarded to two molecular biologists, Emmanuelle Charpentier and Jennifer Doudna for the development of this revolutionary genome editing technique often referred to as “genetic scissors.” The unfortunate aspect of these immunotherapeutic procedures (and CAR-T cell therapies) are their exorbitant costs. Notwithstanding the significant benefits these therapies provide, the costs of FDA-approved CAR-T cell therapy and the CRISPR-Cas9 procedure range from $373,000 to $875,000 for a single treatment. Also, depending on the type of stem cell procedure, prices can range from $5000 to $25,000 per procedure. Gene therapies are subject not only to the regulatory structure of the FDA, but also to the Office of Biotechnology Activities, and the Recombinant DNA Advisory Committee. Excessive regulatory oversight creates an elongated and expensive route to approval. By one estimate, approval for a gene therapy costs nearly $5 billion (five times as much as the average cost of FDA drug approvals). Some insurers are beginning to provide partial coverage of FDA-approved gene therapies, but experimental treatments receive no third-party coverage other than limited humanitarian exemptions. Hopefully, as with other major therapeutic discoveries, the costs of providing the technology will reduce over time.
Finally, a new CRISPR-Cas13 mRNA screen has been developed to establish guide RNAs for the COVID-19 coronavirus and human RNA segments that could be used in vaccines, therapeutics, and diagnostics. Let’s defer a full discussion on this technology to Blog # 36 on infectious diseases, pandemics, and of course, COVID-19.
Discussion Questions:
Treating autoimmune disease includes identifying the “signature” of offending genes (their “gene expression” or the number of RNA molecules they are producing). What technique is used to accomplish this for autoimmune genes?
There now exists gene editing and gene replacement therapies. What is the difference and can you name the gene editing versus the gene replacement technologies?
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