Article Index

The Straight Dope on CRISPR-Cas9 and Cancer

Diana Tamer, PharmD, BCOP
Clinical Assistant Professor
University of Missouri Kansas City School of Pharmacy, Missouri
Hematology/Oncology Pharmacy Specialist
Advent Health Shawnee Mission Cancer CenterKansas City, KS

Thirty years ago, the human genome project began, led by an in­ternational team of researchers looking to sequence and map all genes—together known as the genome.1 Completed in April 2003, it allowed us, for the first time, to read Nature’s complete genetic blueprint for building a human being: around 3 billion DNA base pairs using a four-letter DNA alphabet.1 Subsequent efforts includ­ed profiling patient cancers and exploring germline (inherited) versus somatic (acquired through life) genetic mutations.2 Cancer is a disease of the genome caused by a cell’s acquisition of somatic mutations in key cancer genes, sometimes in addition to inherited germline cancer driving mutations, so these efforts provided great insight into how cancers progress.

Initial cancer genome research focused on protein-coding genes, which together account for approximately 1% of the genome.1 To address this issue, the International Cancer Genome Consortium/ The Cancer Genome Atlas Program (ICGC/TCGA) and the Pan-Can­cer Analysis of Whole Genomes (PCAWG) Project performed whole genome sequencing and integrative analysis on over 2,600 primary cancers. They have now profiled more than 10,000 tumors and generated valuable data that illuminates the complexities of several cancer types.3-5  In 2020, researchers released six papers in Nature and 17 papers in other journals that could pave the way for full ge­nome sequencing of all patient tumors.6 These sequences are being used in efforts to match each patient to a molecular treatment, the hallmark of precision medicine.

In the first part of the 21st century, twin revolutions in biotech­nology and computer science offer enormous promise for technol­ogy to improve our lives. Together, biotech innovations in editing the genome of humans and other organisms, and computer science advancements in machine intelligence and machine learning, have the potential to confer tremendous benefits on humanity. The combination of these two tools could potentially accelerate progress in cancer research dramatically. Various applications could include modelling the genesis and progression of cancer in vitro and in vivo, screening for novel therapeutic targets, conducting functional genomics/epigenomics, and generating targeted cancer therapies.7

Dr. Jennifer Doudna, a professor of chemistry and molecular and cell biology at U.C. Berkeley pioneered the discovery of the fanciest molecular-scissors of the century, which has enabled us to edit DNA and ultimately genomes. Dr. Doudna rocked the research world in 2012 when she and her colleagues announced the discovery of clustered regularly interspaced short palindromic repeats associated nuclease 9 (CRISPR-Cas9); a technology that uses an RNA-guided protein found in bacteria to edit an organism’s DNA quickly and inexpensively.8 In 2020, Dr. Doudna and Dr. Emmanuelle Charpentier, chair of the Regulation in Infection Biology Department at the Helmholtz Centre for Infection Research and a Professor at the Hannover Medical School in Germany, won the Nobel Prize for Chemistry for their work on this powerful gene editing system, increasing awareness of this technology.

What is CRISPR Gene Editing? 9,10
The process of CRISPR has actually existed for millions of years, having evolved to protect bacteria against viruses. The immune systems of certain bacteria use DNA sequences called CRISPR, which contain genetic material collected from viruses to which the bacteria have been exposed. When one of these viruses attacks the bacteria again, the matching CRISPR segment is copied to an RNA molecule that tracks down and binds to the virus’s own DNA, al­lowing a specialized cutting enzyme called Cas protein to chop off a piece of the viral DNA and kill the virus.

Once scientists learned how this worked in bacteria, they were able to extract CRISPR out of bacteria and reprogram the guide RNA to target any DNA sequence of the gene they wanted to alter. That sequence is then attached to a Cas enzyme (molecular “scissors”) to make cuts at the desired locations, adding or removing target DNA. In short, with this technology, we can rewrite the genome. And, this turned out to be simpler, cheaper, more efficient, more precise, and more flexible than previous gene-editing methods.

Somatic gene editing alters DNA of some of the body’s cells in humans to treat genetic conditions. Germline editing manipulates DNA in sperm, eggs, or embryos—affecting all or most-T-cells— and permitting the organism to then pass down those alterations to their offspring. In theory, rather than treating the disease, germline editing could eliminate the disease; and not just from the organism, but from its lineage completely.

A CRISPR Way to Screen for Cancer—A Sci-fi Dream or a Reality? 11-12
Aside from genome editing, CRISPR can also be used to help us rapidly and inexpensively read our DNA. This unexpected finding led to investigating the CRISPR-Cas protein system as a next-gen­eration diagnostic. While Cas9 acts as a precise molecular scissors to produce one cut, Cas12 uses its guide RNA to search billions of letters to find the matching DNA target. Once it does, it starts cutting without stopping just like a paper shredder. Such a protein can be paired with a molecular fluorescent reporter that is ignited when the protein starts shredding and, as a reaction, generates a colorful explosion indicating that the target is present. The reaction detection can be freeze-dried and paper-spotted to generate a visual readout on a lateral-flow test strip, which is cheap and can be used at home, similar to a pregnancy test.

The new diagnostic tool developed by Chen and colleagues could help identify bacterial and viral infections (such as COVID-19), detect cancerous mutations in real time, and recognize new outbreaks before they spread. Cas12 has already been used in vivo to detect the presence of cancer-causing human papillomavirus (HPV) types, a common viral infection that can cause cancers – most commonly cervical cancer. CRISPR-based HPV diagnostics have had almost perfect accuracy. Cas12 can search through fluids such as saliva, blood, or even urine for a specific DNA match in minutes, at the point of care. This has many implications, such as detecting or screening for cancer early, or even diagnosing a viral infection during a pandemic in a prompt, non-invasive fashion.

Moving CRISPR-Cas9 from the Lab to Cancer Patients
The first-in-human testing of CRISPR was in 2016 by Lu and col­leagues in China.13 They performed a Phase I clinical trial to assess the safety of CRISPR/Cas9-mediated knockout of PD-1 gene in autol­ogous T-lymphocyte therapy in patients with metastatic non-small cell lung cancer (NSCLC). The study enrolled 22 patients, 12 of whom were able to receive treatment. Two patients experienced stable disease, no grade 3-5 adverse events were reported, and off-target events were 0.05%. This has been followed by multiple ongoing CRIS­PR trials in China against esophageal, bladder, prostate, renal, and cervical cancers; as well as leukemia and lymphoma.14

The first-in-human CRISPR phase 1 clinical trial in the United States was launched in 2018 by Stadtmauer and colleagues.15 The study was designed to test the safety and feasibility of CRISPR-Cas9 gene editing of T-cells, from patients with advanced refractory cancer. The trial enrolled two myeloma and one liposarcoma patients. They reported observing the edited T-cells expand and bind to tumor targets with no serious side effects related to the investigational approach. These patients were heavily pretreated, and since the trial, one patient has died and the other two have had disease progression. This study was not designed for efficacy, and the number of patients was small. Yet, it represented a historical step in the use of CRISPR-Cas9 in cancer therapeutics.

Improving Current Cancer Treatments with CRISPR
CRISPR may be used to improve efficacy of chimeric antigen receptor (CAR) T-cell therapy.12 Applications under study include the genera­tion of HIV-resistant T-cells with homogenous CAR expression, gener­ation of allogeneic CAR-T-cells, and improving CAR-T cell function.12

Other active areas of study already in clinical trials include improving the efficacy of immunotherapy.23 Unleashing T-cells against tumors by blocking immune checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death 1 ligand 1 (PD-L1) have been successfully used.23 Therefore, the knockdown of these genes using CRISPR, may be crucial to improve the efficacy of immunothera­pies.23 A PD-L1 knockout in mice with ovarian cancer using CRISPR promoted anti-tumor immunity by increasing tumor-infiltrating lymphocytes and modulating cytokine/chemokine profiles within the tumor microenvironment, thus suppressing ovarian cancer progression.24

Future Avenues and Perceived Challenges
Future innovation may include the collaboration of two revolution­ary technologies: CRISPR and artificial intelligence (AI). Machine learning based approaches to examine how changes in our DNA contribute to cancer already exist, and future CRISPR-Cas9 applied cancer therapeutics are likely to reach patients faster using AI to examine potential target and off-target DNA cutting outcomes and their implications.

CRISPR knockdown/knockout models may also offer a promis­ing novel therapeutic approach for cancers that lack effective treat­ments, such as cervical cancer.25 HPV-associated carcinogenesis provides a classical example for CRISPR-based cancer therapies, since the viral oncogenes E6 and E7 are exclusively expressed in cancerous cells.25

Ethical concerns aside, when used for single-gene diseases or cancer, this technology may be the next breakthrough in genetic linked chronic diseases including cancer. Major safety concerns include but are not limited to the unknown long-term consequences of DNA manipulation and the irreversibility of this procedure. Prac­tical and clinical challenges may include side effect management, especially if off-target effects take place; routes of administration; insurance coverage; and affordability. With every new cancer therapeutic modality that is innovated, there is both accompanying promise and peril. Hence, the opportunity for oncology pharma­cists, with their crucial role of collaborating in healthcare teams, to make a difference addressing these concerns.

The effects of innovation are felt around the world. When it comes to medicine, the pace of that change is rapid, especially in oncology, and it’s only moving faster. Remarkable opportunities for good can also be misused. Both malicious intent and unintended consequences can create a real risk of harm for individuals, society, or both.

In 2018, He Jiankui, a Chinese geneticist, claimed to have used CRISPR-Cas9 on a set of twins and a third baby to make them HIV-resistant via editing of their CCR5 gene to create a resistance polymorphism in the children that had previously been seen in nature. His experiments were widely condemned as premature and irresponsible. A commission was formed and on September 3, 2020 the International Commission on the Clinical Use of Human Germline Genome Editing released a 225-page report that offers a guide to the available testing and regulations, as well as the state of current research, and concluded that gene editing of human embryos is not yet reliable enough to use on humans in an ethical way.26  Written by dozens of scientists world-wide, the report stated that any country that permits its scientists to do so should limit the activity to severe, single-gene diseases such as sickle cell anemia, cystic fibrosis, or Tay-Sachs.26

Closing Remarks
Gene editing and AI can radically change cancer therapeutics and is likely to have thousands upon thousands of applications. I believe that this potential for broad and rapid impact is at a scale that has rarely been witnessed in human history. The speed of these changes hastens an already urgent need for discussion on the plans for what to do when there are unmet therapeutic needs and when to proceed with caution especially when it comes to germline gene editing and unknown long-term consequences.

This is an exciting time to be practicing in oncology, witnessing novel therapeutics unfold, and providing new hope for our cancer patients. Yet, it is also a humbling experience that constantly reminds us that we are forever students, and our duty is to pass on this knowledge after we acquire it and consider not only its therapeutic implications, but also its ethical ones. As these tech­nologies are pushed forward, so is our hope to see more scientists in government. There is a need to advocate for more diversity in our representatives to include scientists that can take the lead on these advances, and bridge the gap between science and policy via interdisciplinary collaborations.

Lastly, while using CRISPR in principle to cure sickle cell disease or some cancers may be a dream within reach during our lifetime, it’s not going to do much good if that technology is expensive and remains out-of-reach for the majority of patients. Therefore, the key to moving forward is to take this very exciting development and deploy it in a biomedically ethical, clinically responsible, and patient-affordable way.

Current Clinical Trials Actively Recruiting Using CRISPR in Hematology/Oncology in the United States

Study IdentifierLocationSponsor
A Study of Metastatic Gastrointestinal Cancers Treated with Tumor Infiltrating Lymphocytes in Which the Gene Encoding the Intracellular Immune Checkpoint CISH Is Inhibited Using CRISPR Genetic Engineering – IV infusion16 NCT04426669
  • Masonic Cancer Center, University of Minnesota
Intima Bioscience, Inc.
A Safety and Efficacy Study Evaluating CTX130 (Anti-CD70 Allogeneic CRISPR-Cas9-Engi-neered T-cells) in Subjects with Relapsed or Re-fractory T or B Cell Malignancies – IV infusion17 NCT04502446
  • Duarte, California
  • Houston, Texas
CRISPR Therapeutics AG
A Safety and Efficacy Study Evaluating CTX130 (Allogeneic CRISPR-Cas9-Engineered T-cells) in Subjects with Relapsed or Refractory Renal Cell Carcinoma – IV infusion18 NCT04438083
  • Duarte, California
  • Houston, Texas
  • Salt Lake City, Utah
CRISPR Therapeutics AG
A Safety and Efficacy Study Evaluating CTX001 (Autologous CRISPR-Cas9 Modified CD34+ Hu-man Hematopoietic Stem and Progenitor Cells [hHSPCs]) in Subjects with Transfusion-Depen-dent β-Thalassemia – IV infusion19 NCT03655678
  • Stanford University, California
  • Columbia University, New York
  • The Children’s Hospital at TriStar Centennial Medical Center/ Sarah Cannon Center for Blood Cancers. Nashville, Tennessee
Vertex Pharmaceuticals Incorporated

CRISPR Therapeutics
A Safety and Efficacy Study Evaluating CTX120 (Anti-BCMA Allogeneic CRISPR-Cas9-Engi-neered T-cells) in Subjects with Relapsed or Refractory Multiple Myeloma – IV infusion20 NCT04244656
  • Chicago, Illinois
  • Portland, Oregon
  • Nashville, Tennessee
CRISPR Therapeutics AG
A Safety and Efficacy Study Evaluating CTX110 (Allogeneic CRISPR-Cas9-Engineered T-cells) in Subjects with Relapsed or Refractory B-Cell Malignancies (CARBON) – IV infusion21 NCT04035434
  • University of Chicago – Chicago, Illinois
  • Mayo Clinic – Jacksonville, Florida
  • University of Kansas – Westwood, Kansas
  • Oregon Health and Science University – Portland, Oregon
  • Sarah Cannon Research Institute – Nashville, Tennessee
  • Texas Transplant Institute – San Antonio, Texas
CRISPR Therapeutics AG
A Safety and Efficacy Study Evaluating CTX001 (autologous CD34+ hHSPCs modified with CRISPR-Cas9 at the erythroid lineage-specific enhancer of the BCL11A gene) in Subjects with Severe Sickle Cell Disease – IV infusion22 NCT03745287
  • Lucille Packard Children’s Hospital of Stanford University – Palo Alto, California
  • University of Illinois at Chicago Hospitals and Health Systems – Chicago, Illinois
  • Columbia University Medical Center (21+ years) – New York, New York
  • Children’s Hospital of Philadelphia – Philadelphia, Pennsylvania
  • St. Jude Children’s Research Hospital – Memphis, Tennessee
  • The Children’s Hospital at TriStar Centennial Medical Center/ Sarah Cannon Center for Blood Cancers – Nashville, Tennessee
  • Methodist Children’s Hospital/Texas Transplant Institute – San Antonio, Texas
Vertex Pharmaceuticals Incorporated

CRISPR Therapeutics

The author acknowledges and thank Gerald J. Wyckoff, Ph.D. for review of the article.


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