2020-02-12| Technology

Using the Power of Multiplex Genome Engineering to Treat Cancer

by Ruchi Jhonsa
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By Ruchi Jhonsa, Ph.D.

UPenn researchers report encouraging data from the first US trial that evaluated CRISPR editing in cancer patients

Genome editing is increasingly becoming an attractive option for treating genetic diseases. A variety of genome editing technologies have been developed in the recent past with the intention to make precise genome edits and treat diseases like cancer. One such technology, which has gained much popularity is the CRISPR-based gene editing. This method uses simple Cas9-guide RNA machinery to recognize and either edit or delete mutations in the gene of interest.

Previously used for editing single genetic mutations, CRISPR has now evolved to tackle many genetic changes at once. This new technique termed as Multiplex genome engineering involves modified CRISPR that can perform simultaneous deletion and insertion of multiple DNA sequences in a single round of mutagenesis. In a study recently published in Science, the team of researchers from UPenn, used multiplex genome engineering on T cells in the phase 1 pilot trial to improve adoptive cell therapy in humans. This is the first U.S. clinical trial to test the gene-editing approach in humans and was done in collaboration with Stanford University School of Medicine and was supported by Parker Institute for Cancer Immunotherapy and Tmunity Therapeutics. This is yet another milestone in Penn’s history after being crowned as a pioneer in gene therapy following approval of CAR T cell therapy, Kymriah for pediatric and adult blood cancer patients.

Adoptive Cell Therapy: Similar to CAR T but NOT CAR T

Adoptive cell therapy is based on the idea that T cell receptors (TCR) present on T cells are central for initiating an immune response against tumor cells by recognizing foreign antigens bound to MHC molecules. By altering the T cell receptor for cancer antigens, adoptive cell therapy promises to specifically target foreign cancerous cells. The technology is similar to CAR T in many ways but has some key differences. First, the T cells are extracted from the patient’s body. Next, a transgenic TCR that functions against foreign antigens is expressed in the T cells and reinfused into the patient’s body to combat cancer cells. This technology has been shown to be effective against NY-ESO1 tumor antigen expressed in myeloma, melanoma and sarcoma cancers previously but still poses a few challenges.

Challenges of the Therapy

Technical challenge: Since transgenic TCR’s are expressed in the same cell as endogenous TCR’s, it is a challenge to keep the two molecules apart. Mispairing of endogenous and transgenic TCR has already been reported and is shown to reduce the therapeutic T cell surface expression and activity. Another challenge that the therapy faces is a negative regulatory response from PD-1 protein in response to foreign antigens. It has been observed that removing PD-1 from T cells or masking its function in T cells engineered to attack NY-ESO1 positive cancer cells, increases antitumor efficacy in mice.

Functional challenge: Unlike CAR T which functions independently, TCR modified T cells require the cooperation of a molecule known as HLA-A*02:01, which is only expressed in a subset of patients. Therefore, this method will work in only selective patients.

Phase I Pilot Trial: Testing the Gene Editing Approach

Using multiplex CRISPR editing, endogenous TCR (TRAC and TRBC), as well as PD-1 gene, were deleted in the patient’s T cells producing transgenic TCR against NY-ESO1 antigen. This editing made sure that no mispairing of endogenous and transgenic TCR receptor or negative regulation from PD-1 occurred. Following a short course of chemotherapy, the gene-edited cells were infused into the patients with refractory advanced myeloma and sarcoma and parameters like changes in tumor size, trafficking of modified T cells at the tumor site and adverse effects to the treatment were monitored. Of the three patients that received the therapy, two have progressed and are receiving additional therapies. Of these two patients, one of them showed a 50% reduction in a large abdominal mass. Interestingly, biopsies from bone marrow and tumor showed trafficking of the engineered cells to the sites of tumors in all three patients.

Interesting Observations from the Trial

  1. Infused cells retained all the three edits (deletion of TRAC, TRBC, and PDC-1) even after several months
  2. Engineered cells isolated from the blood of patients several months after the infusion, retained the anti-tumor activity.
  3. The T cells expressing transgenic TCR evolved to a state consistent with central memory in contrast to previous studies where engineered cells evolved to a state consistent with T cell exhaustion.
  4. Modified cells persisted in the blood for as short as 1 month and as long as 9 months. This is a great achievement as previous studies with engineered cells had an initial decay half-life of approximately one week.

Carl June, MD, the Richard W. Vague Professor in Immunotherapy and director of the Center for Cellular Immunotherapies in the Abramson Cancer Center and study’s senior author expressed his joy at the positive outcome of the study and said “This new analysis of the three patients has confirmed that the manufactured cells contained all three edits, providing proof of concept for this approach. This is the first confirmation of the ability of CRISPR/Cas9 technology to target multiple genes at the same time in humans and illustrates the potential to treat many diseases that were previously not able to be treated or cured.”

These new data will open the door to late-stage studies to investigate and extend this approach to broader fields beyond cancer.

Related Article: New ‘Minigene’ Insertion Approach Could Treat Rare Liver Disease in Mice


  2. Stadtmauer et al., 2020, Science
  3. Robbins et al., 2011, J. Clin. Oncol. 29, 917-924
  4. Rapoport et al., 2015, Nat.Med. 21, 914-921
  5. Nowicki et al., 2019, Clin. Cancer Res. 25, 2096-2108
  6. Moon et al., 2016, CLin. Cancer Res. 22, 436-447


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