



Scientists at the Salk Institute have combined CRISPR-Cas9 gene editing with stem cell technology to generate a one-time, autologous cell therapy for the genetic blood clotting disorder hemophilia B. In vivo tests showed that gene-edited, stem cell–derived liver cells remained viable and functional in hemophiliac mice for nearly a year, after just a single injection.

Headed by Suvasini Ramaswamy, Ph.D., and Inder M. Verma, Ph.D., the Salk Institute team's results offer proof of concept for the potential use of autologous cell therapy in the treatment of hemophilia B and potentially other liver disorders that are similarly caused by defects in a single gene. “The appeal of a cell-based approach is that you minimize the number of treatments that a patient needs,” says Dr. Ramaswamy, a former Salk research associate in laboratory of Dr. Verma, and first author of the team’s paper published in Cell Reports. “Rather than constant injections, you can do this in one shot.” The scientists' paper is entitled, “Autologous and Heterologous Cell Therapy for Hemophilia B toward Functional Restoration of Factor IX.”

Hemophilia B is an X-linked clotting disorder that affects 1 in 30,000 male births, the researchers explain. The blood clotting disorder is caused by lack of clotting factor IX (FIX), due to mutations in the FIX gene, and can manifest as either mild, moderate, or severe, dependent upon the extent of FIX activity remaining in patients. Current treatment involves giving patients frequent intravenous doses of recombinant FIX supplements.

Given that hemophilia B is a monogenic disorder, has a broad therapeutic window, and has very good animal models, the disease is “an ideal candidate for gene and/or cell therapy,” the authors note. Gene therapy using adeno-associated viral (AAV) vectors has shown promise for long-term therapy, but viral vector-based approaches carry with them problems, including possible tissue damage and immunogenicity. FIX is produced in the liver, so liver transplantation is an alternative long-term therapeutic option. However, as the team points out, there is a shortage of donor livers and the need for constant immunosuppression represents a major drawback.

Another potential approach is to develop a cell therapy, using cells taken either from donor livers or derived from autologous stem cells. There are three major sources of hepatocytes, the researchers point out—heterologous cadaveric hepatocytes, pluripotent stem cell-derived hepatic-like cells (HLCs) that are derived either from embryonic stem cells (ESCs) or induced pluripotent stem cell (iPSCs), and induced HLCs (Heps) derived by direct reprogramming of fibroblasts into HLCs. Each potential source has its own respective advantages and disadvantages.

In order to test two different approaches to long-term cell therapy, the Salk Institute team first developed a new, quadruple knockout mouse model of hemophilia B that was amenable to the engraftment and expansion of human hepatycytes (hHeps). They first transplanted cadaveric, cryopreserved hHeps, obtained from a range of different vendors, directly into the spleens of the hemophiliac animals. Tests showed that the transplanted cells readily engrafted and remained “healthy, functional and non-tumorigenic” for the duration of the year-long study. Encouragingly, treated animals exhibited sustained increases in levels of human FIX and therapeutic levels of clotting activity. “Depending on the initial number of transplanted cells, anywhere from 10%– 90% of the mouse liver can be humanized by this transplantation and selection approach,” the team writes. “We have tested hepatocytes from multiple donors and sources and have not seen any adverse reactions in the more than 40 animals we have tested so far….We conclude that cadaveric hHeps from heterologous sources produce sustained levels of circulating FIX that can almost completely abolish the clotting defect in our hemophilic mice for up to a year after transplantation (if not longer).”

As an alternative to using heterologous donor hepatocytes, the Salk Institute team developed an approach based on the use of patients' own, gene-corrected and in vitro–differentiated cells. The aim was to generate hepatocyte-like cells (HLCs) from FIX gene-corrected iPSCs derived from peripheral blood-derived mononuclear cells (PBMCs).

First, the team collected blood samples from two severe hemophilia patients and generated iPSCs from the patients' peripheral blood–derived mononuclear cells (PMBCs). They then developed two different CRISPR-Cas9 techniques to correct the relevant gene defect in the iPSCs derived from each patient. The first, more universal approach effectively knocked the correct FIX cDNA into the iPSC’s resident, mutated FIX gene. The second approach involved correcting the missense mutation in the FIX gene, and so restore the original, wild-type gene sequence. In a final step, the Salk team then developed an in vitro–directed differentiation protocol to generate HLCs from both types of the gene-modified iPSC cell lines.

The resulting human HLCs—either with the full FIX gene inserted or with the mutated FIX gene corrected to wild type—were then tested in vitro to confirm that they expressed FIX, and subsequently transplanted into the spleens of the hemophiliac mouse model.

Tests in the recipient animals confirmed that the in vitro–differentiated, patient-derived, gene-corrected iPS-HLCs engrafted and could remain viable and functional over the 10-month study period. Blood samples were analyzed to test for the presence of human albumin (hAlb; a surrogate marker for engraftment efficiency), for FIX, and to test blood clotting ability. Both iPSC-HLC cell lines showed increasing levels of engraftment FIX levels and clotting activity that were “similar to that observed with cadaveric hepatocytes,” the authors write.

The results did indicate that the iPSC-HLC cells didn't engraft as well as cadveric hHEPS, and increases in clotting efficiency in iPSC-HLC recipient animals were “modest,” the authors acknowledge. So, while the iPSC-HLCs did remain functional in the animals’ livers over the long term, “their therapeutic effect could be vastly improved,” the researchers suggest. Encouragingly, data from prior studies of severe hemophilia patients have suggested that even 15% to 20% FIX levels can be enough to stop joint bleeding, “suggesting that even such small repopulation efficiencies by these iPSC-HLCs might be therapeutically active.”

Dr. Ramaswamy acknowledges that “a lot of things have to happen before this can go into humans.” Nevertheless, as the authors state, the study demonstrates the “feasibility of autologous and heterologous cell therapy for treatment of hemophilia B.” They suggest that while “heterologous cell sources such as cadaveric hepatocytes are one alternative, use of autologous iPSC-derived HLCs as a renewable cell source would be ideal.”

The researchers conclude that major benefits of the autologous cell therapy approach include the ability of IPSCs to support homology-directed repair recombination and gene editing. iPSC-derived cells can also be proliferated to support in vitro screening and testing to avoid random integrations and off-target effects, they point out. And because the cell therapy is derived from the patient's own cells, there should be no risk of an immune reaction or the need for long-term immunosuppressive drugs.



























