While many companies use viruses and viral vectors to deliver gene therapy and to modify cells for CAR T treatments, others have shunned adeno-associated viruses (AAV) and lentiviral vectors for other methods to deliver DNA and RNA into the cells.
One of the biggest problems with AAV is the insert size is limited to about 4.7 kilobases (kb).
"Some of the gene therapeutic products and candidates have truncated the size of the genes and have done some pretty clever maneuvers to try and make the gene inserts smaller in order to be incorporated by AAV," Daniel Dornbusch, head of business development at Dnarx Inc., explained, while noting that the full length might be better and there are many genes that can't be truncated.
The use of viruses also causes immune reactions that can limit the re-treatment of patients who will develop antibodies to the virus. And many patients are immune to an initial treatment simply because they've previously been naturally exposed to the virus or a related one.
"You have one shot at it and that's it," Dornbusch said of gene therapy.
AAVs are better for gene therapy than first-generation gene therapy constructs that were oncogenic because they integrated randomly into the genome. AAV-delivered vectors, on the other hand, exist as an episome. But the downside to the lack of integration is that the expression can be weak.
"Right now almost all diseases that are being treated with AAV – you only need a small percent of wild-type in order to correct the disease," Eric Ostertag, CEO of Poseida Therapeutics Inc., told BioWorld. "That problem is compounded when you go to a disease that requires 50% of wild-type levels – you simply can't do that with AAV currently – or if you're trying to treat a tissue like the pediatric liver, where it's a rapidly dividing tissue, that will very quickly dilute your non-integrating AAV to zero."
To overcome the challenges of AAV, newco Dnarx developed the High-level Extended Duration Gene Expression System (HEDGES), which consists of a DNA vector and a modified lipid carrier.
In model systems, San Francisco-based Dnarx has been able to get DNA as large as 30 kb into cells, and it hasn't found the upper limit on size the system can accommodate. The added space allows room for regulatory elements or even multiple genes. Dnarx has expressed six genes through one construct.
The makeup of the lipid carrier allows Dnarx to vary the duration of expression from less than three weeks to more than 1.5 years, or perhaps even longer in humans.
Last week, Dnarx announced a $10.7 million contract with the Defense Advanced Research Projects Agency (DARPA) to develop DNA-encoded gene-based therapeutics to protect against pandemic influenza.
DARPA wants Dnarx to use HEDGES to express a broadly neutralizing antibody, which could result in protection from a pandemic influenza in a day, compared to weeks that it can take for the immune system to produce antibodies to an antigen-based vaccine.
The project will also explore the use of dCas, a CRISPR-based system that doesn't cut the DNA but can up- or down-regulate genes that it binds to. For DARPA, Dnarx would use HEDGES to deliver a dCas capable of up-regulating genes that protect humans from infections.
Beyond influenza, Dnarx has preclinical programs to express monoclonal antibodies, such as rituximab – Roche Holding AG and Biogen Inc.'s anti-CD20 antibody Rituxan – and mepolizumab, which Glaxosmithkline plc developed as Nucala for asthma. By expressing antibodies in the patient, dosing could be extended from every couple of weeks to every few years.
Dnarx is also using HEDGES for expressing proteins, such as factor VIII for hemophilia and G-CSF for cancer, and the company has some undisclosed programs for rare diseases.
Selecting expressed cells
San Diego-based Poseida has developed Piggybac, a system that uses a transposon with inverted terminal repeat sequences flanking the DNA to be delivered and a transposase enzyme that cuts the DNA out of the transposon and integrates it into the genome.
The larger size allows Poseida to include multiple CAR targeting genes in its CAR T constructs. Its constructs also include a safety switch to eliminate the T cells if cytokine release syndrome occurs and a drug resistance gene to select cells that have incorporated the CAR.
"With the viral approach, you get a certain amount of transduction on the front end, and there's some percent of your cells that are not genetically modified. And you're just putting those back in and those cells are taking up slots that would otherwise be occupied, ideally, by genetically modified cells," Ostertag explained. "What we do is the positive selection, so 100%, essentially, of our final product is genetically modified."
Piggybac also preferentially integrates into less differentiated stem memory T cells (T-SCM), which should increase the duration of the response since T-SCM cells can survive for decades as they create new killer T cells that attack the cancer.
Not having to use viruses speeds up the manufacturing time and decreases the cost, which Ostertag estimates may be $50,00 to $60,000 cheaper per manufacturing run.
Poseida's lead CAR T, which targets BCMA, is being tested in a phase I study, and the company plans to file an IND for its next CAR targeting PSMA expressed on prostate cancer later this year. Further back, there's an allogeneic CAR T targeting BCMA, which should begin clinical development next year. And Poseida is in the early stages of developing dual CAR products that express CARs honing in on multiple targets.
Maxcyte Inc. uses flow electroporation to get DNA and RNA into cells, which decreases the manufacturing time. The system can treat 1 x 10^10 cells in 20 minutes, allowing a run to be done in less than a day, compared to five to 10 days for DNA delivered with viruses.
The Gaithersburg, Md.-based company has licensed its flow electroporation technology for more than 80 programs, including around 30 for clinical purposes.
Maxcyte is also using the technique to develop its Carma platform that uses non-expanded fresh T cells expressing a CAR from a delivered mRNA. The transient expression from an mRNA, which will eventually be degraded, makes them safer, but requires multiple treatments.
Fortunately, the quicker manufacturing makes it easier to deliver multiple doses – a single manufacturing run can produce six to 20 treatments – and there could be some advantages to re-treating.
"With solid tumors, the tumor environment is very immuno-tolerant. What you need to do is break that tolerance," Doug Doerfler, president and CEO of Maxcyte, said. "Dosing multiple times will break the tolerance and reactivate the immune system and eventually create an immune cascade against those cancerous cells."
Maxcyte's lead molecule, MCY-M11, which targets mesothelin, is in phase I development for patients with peritoneal mesothelioma or adenocarcinoma of the ovary, primary peritoneum or fallopian tube.