Gene Correction of Autologous Hematopoietic Stem Cells in Artemis Deficient SCID

Children born with Severe Combined Immunodeficiency Disease (SCID) are unable to fight common infections and often die within the first 6 months of life. The most severe form of SCID is the result of an inherited deficiency of an essential chemical in the body called Artemis (SCID-A) without which T and B cells are unable to develop. Artemis is an essential component of a process which repairs DNA (the instructions in cells which make us who we are) when it is damaged or cut during normal metabolic activities. Because of this, children with SCID-A are also more sensitive to typical chemotherapeutic drugs used for bone marrow stem cell transplant (BMT). For these reasons, if the patient’s own blood stem cells could be corrected by inserting a normal human Artemis gene into their DNA and these cells put back into the patient’s bone marrow to grow and restore normal T and B lymphocyte function, many if not all of the problems associated with BMT would be eliminated. In order to prove that the viral vector that we have constructed for clinical use this past year, that contains the human gene for Artemis, can effectively insert the Artemis gene into mouse and human cells we are using a variety of assays. We have shown that the Artemis protein is made by the gene when Artemis-deficient skin cells are "infected" with this viral vector and that these cells which previously were susceptible to radiation are now similar to normal cells. We have also established that normal human stem cells can be transduced (or "infected") with a control(non-Artemis)vector and still mature into human T cells (in mice) and when cultured outside the body on a special "feeder" layer of cells that support maturation of stem cells into B cells. We plan to repeat these experiments with our "clinical grade" viral vector using normal and Artemis-deficient human and mouse stem cells. We are also studying ways that we can open up "space" in the bone marrow to permit large numbers of donor stem cells to live and grow. For a typical bone marrow transplant this is done by pre-treating the patient (recipient) with high doses of chemotherapy that destroy the patient's own stem cells and allow the donor stem cells to stay and grow in the bone marrow. Since patients (and mice) with Artemis deficiency are unable to tolerate these high doses of chemotherapy, we are studying approaches that don't require this pre-treatment. In early experiments we have demonstrated that a drug called anti-ckit (ACK2) that attacks bone marrow stem cells in severely immunodeficient mice, also works in Artemis-deficient mice and following transplantation of highly purified normal mouse stem cells, the ACK2-treated mice begin to generate both donor T and B lymphocytes that are absent in Artemis deficiency as early as 4 weeks post transplant. The ACK2 is not a chemotherapy drug and has no other toxicity than its affect on bone marrow cells. Now that we have the clinically comparable viral vector we are planning experiments with Artemis deficient donor stem cells that will be treated with the vector so that they are no longer Artemis-deficient. With ACK2 pre-treatment we expect that these cells will grow in the bone marrow of Artemis-deficient mice and restore normal immune function. There is a preparation of ACK2 that may be used in humans but this will require further study. Therefore, we plan in the coming year to evaluate three other possible agents that are currently available for use in humans and that may also be effective in opening "space" in the marrow and allow the viral vector-treated bone marrow stem cells to grow and mature.

Children born with Severe Combined Immunodeficiency Disease (SCID) are unable to fight common infections and often die within the first 6 months of life. The most severe form of SCID is the result of an inherited deficiency of an essential chemical in the body called Artemis (SCID-A) without which T and B cells are unable to develop. Artemis is an essential component of a process which repairs DNA (the instructions in cells which make us who we are) when it is damaged or cut during normal metabolic activities. Because of this, children with SCID-A are also more sensitive to typical chemotherapeutic drugs used for bone marrow stem cell transplant (BMT). For these reasons, if the patient’s own blood stem cells could be corrected by inserting a normal human Artemis gene into their DNA and these cells put back into the patient’s bone marrow to grow and restore normal T and B lymphocyte function, many if not all of the problems associated with BMT would be eliminated. We have now shown that the viral vector that we have constructed for clinical use, that contains the human gene for Artemis, can effectively insert the Artemis gene into mouse and human cells. We have shown that the Artemis protein is made by the gene when Artemis-deficient skin cells are "infected" with this viral vector and that these cells which previously were susceptible to radiation are now similar to normal cells. We have also established that our Artemis vector can correct Artemis-deficient human and mouse stem cells. We are also studying ways that we can open up "space" in the bone marrow to permit large numbers of donor stem cells to live and grow. For a typical bone marrow transplant this is done by pre-treating the patient (recipient) with high doses of chemotherapy that destroy the patient's own stem cells and allow the donor stem cells to stay and grow in the bone marrow. Since patients (and mice) with Artemis deficiency are unable to tolerate these high doses of chemotherapy, we are studying approaches that don't require this pre-treatment. We have demonstrated that a drug called anti-ckit (ACK2) that attacks bone marrow stem cells in severely immunodeficient mice, also works in Artemis-deficient mice and following transplantation of highly purified normal mouse stem cells, the ACK2-treated mice begin to generate both donor T and B lymphocytes that are absent in Artemis deficiency as early as 4 weeks post transplant. The ACK2 is not a chemotherapy drug and has no other toxicity than its affect on bone marrow cells. We have also started evaluating very low doses of chemotherapy to identify the minimal dose that will still be effective in restoring immune function in our SCID mice. In the coming year, we plan to evaluate three other possible agents that are currently available for use in humans and that may also be effective in opening "space" in the marrow and allow the viral vector-treated bone marrow stem cells to grow and mature. Finally, we plan to evaluate possible harmful effects that our vector might cause and have begun discussions with the FDA to plan a clinical trial in humans with Artemis deficiency.

Children born with Severe Combined Immunodeficiency Disease (SCID) are unable to fight common infections and often die within the first 6 months of life. The most severe form of SCID is the result of an inherited deficiency of an essential chemical in the body called Artemis (ART-SCID) without which T and B cells are unable to develop. Artemis is an essential component of a process which repairs DNA (the instructions in cells which make us who we are) when it is damaged or cut during normal metabolic activities. Because of this, children with ART-SCID are also more sensitive to typical chemotherapeutic drugs used for bone marrow stem cell transplantation (BMT). For these reasons, if the patient's own blood stem cells could be corrected by inserting a normal human Artemis gene into their DNA and these cells put back into the patient's bone marrow to grow and restore normal T and B lymphocyte function, many if not all of the problems associated with BMT would be eliminated. We have now shown that the viral vector that we have constructed for clinical use, that contains the human gene for Artemis, can effectively insert the Artemis gene into mouse and human cells. We have shown that the Artemis protein is made by the gene when Artemis-deficient skin cells are "infected' with this viral vector and that these cells which previously were susceptible to radiation are now similar to normal cells. We have also established that our Artemis vector can correct Artemis-deficient human and mouse stem cells. We are also studying ways that we can open up "space' in the bone marrow to permit large numbers of donor stem cells to live and grow. For a typical bone marrow transplant this is done by pre-treating the patient (recipient) with high doses of chemotherapy that destroy the patient's own stem cells and allow the donor stem cells to stay and grow in the bone marrow. Patients (and mice) with Artemis deficiency are unable to tolerate these high doses of chemotherapy. We have shown that very low doses of this chemotherapy can be used in ART-SCID mice in order to restore immune function and that these doses are ineffective in normal mice indicating that ART-SCID bone marrow cells are more sensitive that normal cells to chemotherapy. We have also evaluated the potential harmful effects that too much Artemis protein might have on bone marrow stem cells and have identified the optimal dose of Artemis vector that minimizes toxicity. We have shown that this vector does not appear to alter normal genes when inserted into normal cells. Finally, we have developed a clinical protocol for evaluating the vector in a first in human clinical trial and have met with the FDA to discuss what we must do in order to apply for an IND for this clinical trial. In the coming year we plan to complete these IND-enabling studies.

We have met almost all of the milestones and goals of this project. We have documented the efficacy of the Artemis vector in Artemis-deficient bone marrow stem cells by demonstrating that they are capable of maturing into T and B cells. We have also identified the optimal conditions for correcting the Artemis defect in these cells. Finally, we have demonstrated the safety of the vector and have shown that we can correct the defect in Artemis deficient mice.