Introduction: It is well known that in vivo gene editing can treat disease-causing genes or insert functional genes through gene editing of target cells in vivo to treat diseases that were previously considered incurable, such as genetic diseases and cancer. In 2020, CRISPR gene editing technology won the Nobel Prize in Chemistry; on June 27, 2021, the first clinical data on the safety and efficacy of CRISPR gene editing in vivo was published at NEJM, showing that a single intravenous injection of CRISPR can accurately edit target cells in vivo and treat genetic diseases.
Gene-editing technology can radically treat cancer
Gene editing techniques are mostly used in gene function research and disease treatment, and common tools include Zinc Finger Endonuclease (ZFN), Transcription Activator Like Effector Nuclease (TALEN), and CRISPR/Cas (Clustered Regularly Interspaced). Short Palindromic Repeats-associated) system (Table 1).
Table 1 Comparison of three gene editing techniques
Source: [1] 丨 Tabulation: Bio-Quest Editorial Team
The CRISPR/Cas system is widely present in the natural immune system of bacteria and most archaea and consists mainly of two parts: the gene encoding the Cas protein and the CRISPR sequence consisting of a leading sequence, discontinuous repeat sequences, and sequences of similar intervals. Applications include: the region to be edited has conservative PAM sequences (Proto-spacer Adjacent Motifs), sgRNAs that can be paired complementarily with the upstream PAM sequence, and functional Cas enzymes. Compared with traditional gene editing tools, the CRISPR/Cas system has outstanding advantages.
According to the different effector proteins, CRISPR/Cas systems are divided into two categories (Figure 1): Class 1 systems are complexes composed of a variety of different effector proteins, usually forming multi-subunit protein crRNA (CRISPR RNA) effector complexes, including I, III. and IV. and 12 subtypes; 2 types of systems only have a single effector protein (Cas9, Cas12, Cas13) to interfere with the gene of interest, accounting for about 10% of the CRISPR/Cas system, including II, V. and VI. and 9 subtypes, in addition to cutting DNA, Cas13a (C2c2) in class 2 systems can target ssRNA (Single-stranded Ribonucleic Acid).
Fig. 1 Classification of CRISPR/Cas systems (Source: [1])
The mechanism of action of the CRISPR/Cas system is divided into three stages (Figure 2).
(1) Adaptation: Ingestion of exogenous genetic material
After the invasion of exogenous genetic material, the CRISPR/Cas system recognizes the PAM sequence of the exogenous gene and obtains part of the exogenous fragments from near the PAM sequence to form an interval sequence, integrating from the 5' end to the CRISPR repeat sequence so that it has a "memory" that can specifically destroy the invading nucleic acid at the time of the next infection, and most known CRISPR/Cas systems require the participation of the Cas1-Cas2 complex in this process.
(2) Expression: Expression and maturation of crRNA
The CRISPR region is first transcribed to pre-crRNA, then cleaved into mature crRNAs containing 1 spacer sequence and partially repeated sequences, directly or further processed to bind to Cas proteins into "effectors" or "interference" complexes with specific nucleic acid endonuclease activity.
(3) Interference: shearing of exogenous genetic material
The complex is scanned along the exogenous genetic material under the guidance of crRNA, and when there is a crRNA matching region near the PAM sequence, the effector complex is cleaved to promote the degradation of nucleic acid molecules, and the exogenous nucleic acid expression is silent.
Cas9 belongs to the Class 2 Type II CRISPR system, which has been the most widely used genome editing tool since it was validated in human cells in 2013, and in order to make it have a greater scope of action, the development of this tool is also deepening.
Fig. 2 Schematic diagram of CRISPR/Cas mechanism (Source: [1])
Tumors are formed by the accumulation of somatic mutations, and there are different mutation genes and mutation sites depending on the type of cancer. Traditional cancer treatment methods have limited effect and high recurrence, and inactivating oncogenes or activating tumor suppressor genes through gene editing technology, and then changing downstream signaling pathways for cancer treatment, can fundamentally treat diseases. As a gene editing tool, CRISPR/Cas9 can be used for homologous directed repair, gene knockout, insertion, chromosomal ectopicity, chromatin recombination, disease diagnosis and other work. At present, CRISPR/Cas9 technology has been applied to basic research (Table 2) and biological therapy (Table 3) in tumor treatment.
Table 2 Basic research on CRISPR/Cas9 in tumor treatment
Table 3 CRISPR/Cas9-mediated tumor biotherapy
CRISPR technology has outstanding advantages and clinical applications
On 17 June 2021, The New England Journal of Medicine (NEJM) reported the world's first clinical data supporting the safety and efficacy of CRISPR gene editing in vivo (Figure 3). The study was conducted by teams from the National Centre for AmyloidOsis, The University of London, St George's University, Royal Free College, the University of Auckland and New Zealand Clinical Research, and co-sponsored by Intellia Therapeutics and Regeneron.
Figure 3 Research results (Source: NEJM)
Transthyroxine amyloidosis (ATTR) is a life-threatening disease characterized by misfolded transthyroxine protein (TTR) proteins that accumulate sexually mainly in the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent designed to treat ATTR amyloidosis by reducing the concentration of TTR in the serum. NTLA-2001 is based on a clustered regular interval of short palindromic repeats and a related Cas9 nucleic acid endonuclease (CRISPR-Cas9) system that encapsulates messenger RNA of Cas9 proteins by lipid nanoparticles and uniguid RNA that targets TTR (Figure 4).
Figure 4 Mechanism of action of NTLA-2001 (Source: NEJM)
The interim clinical data published in the study covered 6 ATTR patients treated in the Phase 1 clinical trial, of which 3 received NTLA-2001 at a dose of 0.1 mg/kg and 3 received a dose of 0.3 mg/kg. The following results were obtained by measuring the TTR concentration level in the patient's serum, the pharmacology of NTLA-2001 in vivo, the in vitro evaluation of NTLA-2001, and the evaluation of adverse events and off-target effects:
1. On the 28th day of receiving NTLA-2001 treatment, a single dose of NTLA-2001 can reduce the TTR level in the patient's serum depending on the dose. TTR levels decreased by an average of 52% in the 0.1 mg/kg dose group and 87% in the 0.3 mg/kg dose group, with one patient having a 96% decrease in TTR levels.
2. On the 28th day of receiving NTLA-2001 treatment, NTLA-2001 showed good safety, and no serious adverse events and liver problems were found. All adverse events were mild (Grade 1);
3. The therapeutic dose of NTLA-2001 did not produce an "off-target effect".
Dr. John Leonard, President and CEO of Intellia, funder of the research, said: "For the first time ever, in vivo gene editing clinical data show that a single intravenous infusion of the CRISPR system can accurately edit target cells in patients to treat genetic diseases. The interim results of NTLA-2001 validated the hypothesis that it had the potential to abort and reverse ATTR in the case of a single dose. Solving the challenge of targeted delivery of the CRISPR/Cas9 system to the liver and also opening the door to the treatment of other genetic diseases based on our technology platform, we plan to rapidly advance and expand our R&D pipeline." These data make us believe that we are truly ushering in a new era of medicine. ”
Google Venture Capital is bullish on CRISPR technology and has added positions to startups
On March 22, 2022, Spotlight Therapeutics ("Spotlight"), which was founded in 2017, announced that it had raised $36.5 million in Series B funding, resulting in a total of $80.7 million in Funding from Spotlight. The round was co-led by GordonMD Global Investments and EPIQ Capital Group, with participation from Magnetic Ventures and existing investors such as GV (formerly Google Ventures) and Emerson Collective (Table 4).
Table 4 Spotlight Therapeutics financing information
Source: [3] 丨 Tabulation: Bio-Exploration Editorial Team
Spotlight is committed to developing in vivo CRISPR gene editing therapies and has a proprietary technology platform that targets the Targeted Active Gene Editors (TAGE) platform. The TAGE platform "partsize/modularize" cell-penetrating peptides (CPPs), ligands, and antibodies to target cell categories, as well as protomoles of multiple nucleases, and establish a library of parts to combine these "parts"/"modules" to achieve optimal cell selectivity and efficacy of molecules. This "parted/modular" approach avoids the complexity and toxicity of current cell, viral, and nanocarrier delivery methods (Figure 5).
Figure 5 TAGE platform (Source: Spotlight Therapeutics official website)
TAGE is able to target selected cell types in vivo. This has the potential to improve treatment effectiveness and enable editing of cell types that are not easily targeted by current delivery methods. TAGE's "parted/modular" configuration allows the creation of "fit-for-purpose" molecules by altering the cell targeting section, CRISPR effectors, and gRNA. At the same time, TAGE has a short half-life, so it does not persist in the body after completing the task. This reduces the risk of off-target, minimizes the likelihood of an anti-drug immune response, and provides greater flexibility in dose.
Dr Mary Haak-Frendscho, President and CEO of Spotlight, said: "This Series B funding round is an important milestone that will allow us to advance the immuno-oncology (IO) project (Figure 6), as well as projects on ophthalmic diseases and hemoglobinopathies. Will allow us to harness Spotlight's unique cell-targeted in vivo delivery approach to unlock the full potential of gene editing, providing patients with effective one-off therapeutic drugs." ”
Figure 6 IO project (Source: Spotlight Therapeutics official website)
The CRISPR/Cas9 system can break through the limitations of traditional diagnosis and treatment methods and is a revolutionary cancer treatment method. It is believed that with the continuous deepening of research, CRISPR/Cas9 will play a greater role in basic tumor research and clinical applications.
Source: Spotlight Therapeutics official website, for academic exchange only.
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Resources:
[MA Mengdan,YANG Yubin,CHEN Yanping,et al.CRISPR/Cas9 technology and its application in tumor research and treatment[J]Life Sciences,2021,33(11):1370-1381.DOI:10.13376/j.cbls/2021153.
[2] Gillmore JD, Gane E, Taubel J, et al. CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. N Engl J Med. 2021 Aug 5;385(6):493-502. doi:10.1056/NEJMoa2107454. Epub 2021 Jun 26. PMID: 34215024.
[3]https://www.crunchbase.com/organization/spotlight-therapeutics/company_financials
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