While gene editing technologies have been blowing up over the past few years, another gene regulating technology has also been gaining speed: RNA interference (RNAi). RNAi works by interfering with gene expression by binding to the gene’s corresponding mRNA molecule, rather than physically changing the DNA sequence like gene editors such as CRISPR do. Unlike gene editing, RNAi is not a complete “on/off” switch because it doesn’t permanently alter the genetic code, possibly allowing a small amount of gene expression.
Other than being explored as a novel therapeutic pathway, RNAi can be used to alter gene expression in cells and create cellular therapies, such as CAR-T therapy.
CAR-T Therapy Basics and Other Allogeneic CAR-Ts in Development
CAR-T cells are T-cells that have been engineered to express a receptor (called a chimeric antigen receptor, or CAR) that recognizes a specific type of cancer cell. These altered T-cells can now recognize and attack cancer cells specifically. Cell engineering to create CAR-T cells is commonly accomplished by adding the desired DNA into cells using a viral vector, but it can also be achieved through gene editing and RNAi.
Currently approved CAR-T therapies are autologous, or patient-specific, so they cannot be used for multiple patients. The next major hurdle will be getting an allogeneic, or “off-the-shelf,” CAR-T therapy approved. Allogeneic CAR-T cells are ideal because they can be created from healthy donor cells, engineered to express the desired CAR, then used on any patient.
Most other allogeneic CAR-T therapies involve gene editing with either CRISPR or transcription activator-like effector nucleases (TALENs). Cellectis is developing four allogeneic (or “universal”) CAR-T therapies: UCART123, for treating CD123+ leukemic cells such as those in the majority of acute myeloid leukemia (AML) patients, which is currently in a Phase 1 trial for adult AML patients; UCART22, TALEN gene-edited CAR-T cells for treating CD22+ acute lymphoblastic leukemia (ALL) precursor B-cells, which is currently in a Phase 1 trial for adult B-cell ALL patients; UCARTCS1, gene-edited CAR-T cell for treating CS1+ hematologic malignancies such as multiple myeloma (MM), which has been IND-approved by the FDA to begin clinical trials; and UCARTCLL1, gene-edited CAR-T cells for treating CLL1+ hematologic malignancies such as AML, which is in pre-clinical development.
Cellectis, Allogene and Servier are also jointly developing two allogeneic CAR-T therapies: UCART19, TALEN gene-edited CAR-T cells for treating CD19+ hematologic malignancies such as ALL, which is in Phase 1 trials for pediatric ALL patients (PALL) and adult ALL patients (CALM); and ALLO-501, gene-edited CAR-T cells for treating CD19+ non-Hodgkin lymphoma (NHL), which is in a Phase 1 trial for adult NHL patients. Cellectis and Allogene are also working on two other allogeneic CAR-T therapies: ALLO-715, gene-edited CAR-T cells for BCMA+ MM, which is currently in pre-clinical development; and ALLO-819, gene-edited CAR-T cells for treating FLT3-expressing AML, which is in pre-clinical development.
Allogene is also developing a few other allogeneic CAR-T therapies: CD70, which is in pre-clinical development for both hematologic malignancies and renal cell carcinoma (RCC); DLL3, which is in pre-clinical development for small cell lung cancer (SCLC); and ALLO-647, an anti-CD52 monoclonal antibody used as a lymphodepletion agent (a drug that reduces the amount of lymphocytes, which mediate transplant rejection, in an effort to prevent rejection) in the Phase 1 study of ALLO-501 in adult NHL patients.
CRISPR Therapeutics is also developing three allogeneic CRISPR/Cas9 gene-edited CAR-T therapies, which include CTX110 for CD19-expressing B-cell malignancies, which is in pre-clinical IND-enabling development; CTX 120 for BCMA-expressing MM, which is in pre-clinical IND-enabling development; and CTX130 for CD70-expressing hematologic malignancies (such as some lymphomas) and solid tumors (such as RCC), which is in pre-clinical development.
Using RNAi to Create Off-the-Shelf CAR-T Therapy
Now, the biotech company Celyad is looking for a non-gene-edited approach to create allogeneic CAR-T cells using RNAi. Last October, Celyad announced a collaboration with shRNA experts Dharmacon, a Horizon Discovery company, to use their optimized shRNA technology to create CAR-T therapies.
To make a successful allogeneic CAR-T therapy, you must ensure that the donor CAR-T cells won’t non-specifically react with the healthy host cells. Celyad employs various methods, including shRNA, to knock down the T-cell receptor (TCR) complex in T-cells by blocking the expression of a particular protein in the complex called CD3-zeta. The TCR complex is made up of eight proteins that group together on the T-cell surface and are important to a T-cell’s function. Without the CD3-zeta protein, the TCR complex isn’t expressed properly on the T-cell surface and can’t properly transmit signals. This means that the donor-derived modified T-cells shouldn’t attack the host’s own cells, a complication known as graft-versus-host disease (GvHD).
According to Filippo Petti, CEO of Celyad, most gene editing focuses on another component of the TCR complex called the TCR alpha chain. Although shRNA can be used to knockdown multiple components of the TCR complex, Celyad chose to focus on blocking CD3-zeta expression because it is the “rate-limiting step” in TCR complex expression. Petti said that they achieved TCR complex knockdown using shRNA comparable to that using CRISPR/Cas9 gene suppression.
Celyad engineers T-cells to express a CAR incorporating the NKG2D receptor, which recognizes certain stress markers that are overexpressed on many types of cancerous cells. Initially, they have focused on adult acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and multiple myeloma (MM).
To get all the necessary components into the T-cells, they use an all-in-one vector that encodes the CAR, shRNA, and a tag that allows for positive selection of cells that express both components (the CAR and shRNA). This screening methodology is unique as only negative selection can be done on gene edited-CAR-T cells to verify that the desired gene was knocked out. Having both a positive screen (via expression of the vector-encoded tag) and a negative screen (via TCR complex knockdown) allows Celyad to efficiently select the correct CAR-T cells.
The “SMARTvector” platform was developed and optimized by Dharmacon, who screened naturally occurring human microRNA to select a scaffold that not only allows for efficient processing of the shRNA from the vector and the expression of all the vector components, but also doesn’t overexpress the antisense shRNA, thereby avoiding cellular toxicity.
How Could Non-Gene Edited-CAR-T Therapies Make a Difference in the Clinic?
In mice, both gene-edited and shRNA-modified CAR-T cells prevented major weight loss, a typical characteristic of GvHD, and showed similar rates of survival.
Interestingly, the CAR-T cells created using shRNA persisted longer than those created using CRISPR/Cas9. Petti hypothesized that, because shRNA isn’t a strict “on-off” switch, shRNA-modified T-cells could still allow some TCR complex signaling. This would keep the T-cells “happy, persisting, and replicating – keeping the T-cells behaving like T-cells,” rather than completely shutting down signaling like gene editing does. It’s important to note that enough TCR complex signaling is shutdown to decrease the likelihood of GvHD in mice.
Pairing this longer persistence (and potentially longer duration of effect) with being created using a single all-in-one vector and positively selecting the properly expressing cells allows for shRNA-created CAR-T cells to be easier and cheaper to produce than CRISPR gene edited CAR-T cells.
“shRNA can be a disruptor for two reasons: shRNA-modified CAR-T cells are more persistent than gene-edited CAR-T cells, which could possibly be beneficial in the clinic and the fact that shRNA has an established history,” Petti told BioSpace. “RNAi has been around for about 20 years, it has been optimized, and there is already other RNAi technology being studied and available.”
Celyad’s CAR-T Therapies in Development
Their lead candidate CYAD-01, an autologous CAR-T therapy using their NKG2D CAR technology, has already been shown to be well-tolerated in ongoing Phase 1/2 (THINK), Phase 1 (DEPLETHINK) and Phase 1 (SHRINK) trials for the treatment of both hematologic malignancies (AML, MDS and MM) and solid tumors (colorectal, ovarian, and pancreatic cancers).
These results are positive as Celyad is developing other CAR-T therapies based on this NKG2D CAR technology, such as CYAD-101, an allogeneic CAR-T therapy utilizing their TCR inhibiting molecule (TIM) to disrupt TCR complex signaling. TIM is a truncated form of the CD3-zeta protein, which interferes with TCR complex signaling. It is an alternative approach to shRNA that prevents donor CAR-T cells from reacting with the host cells, ultimately preventing unwanted serious side effects like GvHD. CYAD-101 is currently being studied in an open-label Phase 1 clinical study (alloSHRINK) for 36 patients with metastatic colorectal cancer.
Celyad is also developing the CYAD-200 series of novel, non-gene edited shRNA-based CAR-T therapies. CYAD-200 therapies are created using the “SMARTvector” technology to express a CAR and shRNA targeting CD3-zeta to knockdown the TCR complex.
For the 200 series, Celyad is developing three first-in-class allogeneic non-gene edited CAR-T therapies: CYAD-211 targeting B-cell maturation antigen (BCMA) to treat MM (expected to enter the clinic in mid-2020); CYAD-221 targeting CD19 to treat B-cell malignancies (expected to enter the clinic in late 2020); and CYAD-231 targeting both NKG2D and an undisclosed membrane protein to treat an undisclosed disease (expected to enter the clinic in early 2021).