TKID gene therapy: a new success

Lentiviral vector cured children of congenital immunodeficiency

Vyacheslav Gomenyuk, N+1

American and British scientists using the lentiviral vector ex vivo launched the synthesis of adenosine deaminase in children with insufficiency of this enzyme in blood stem cells and, thus, cured congenital immunodeficiency caused by this defect. As reported in The New England Journal of Medicine (Kohn et al., Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency), in several studies, two- and three-year survival was 100 percent.

Adenosine Deaminase (ADA) – it is an enzyme of purine metabolism that catalyzes the deamination of adenosine to form inosine, deoxyinosine and deoxyadenosine. Deoxyadenosine is a toxic compound, therefore, with a lack of ADA, it accumulates in lymphocytes, disrupts DNA synthesis and leads to violations of both (cellular and humoral) links of immunity, causing severe congenital combined immunodeficiency. Very few lymphocytes are found in the blood of such children, which is why they are at extremely high risk of infection and, on average, live up to two years.

It is possible to completely cure the deficiency of ADA with the help of hematopoietic stem cell transplantation. However, scientists and doctors are trying to invent more gentle methods of treatment, since transplantation is associated with other risks, including infectious ones. For example, such patients are initially prescribed replacement therapy with bovine adenosine deaminase modified by covalent addition of an inert polymer of polyethylene glycol. However, it does not provide a complete restoration of immunity.

Doctors have already tried to cure this pathology of immunity with the help of genetically modified drugs. In 2016, the European Medical Agency approved gene therapy with autologous hematopoietic stem cells, which were modified ex vivo using a gamma retroviral vector. However, in clinical trials involving patients with primary immunodeficiency, the use of γ-retroviral vectors led to vector-related leukemias and myelodysplastic diseases. Recently, a patient was diagnosed with lymphoid T-cell leukemia 4.7 years after such gene therapy.

A group of scientists led by Donald B. Kohn from University of California and Claire Booth from University College London has developed a self-inactivating lentiviral vector EFS-ADA LV, in which scientists removed an enhancer promoter with a long terminal repeat, and instead inserted a "shortened" promoter of the human elongation factor 1a gene to stimulate the expression of the codon-optimized complementary DNA transgene ADA. This promoter has a significantly lower transactivation potential than gamma-retroviral promoters.

In the present study, scientists evaluated the safety and efficacy of the studied lentiviral gene therapy, which consists of the patient's own CD34+ hematopoietic stem and progenitor cells that were genetically modified ex vivo using a lentiviral vector.

A total of 50 patients with ADA insufficiency received gene therapy in three studies – 30 in the United States (average age 10 months) and 20 in the United Kingdom (10 people included in the study and 10 people treated under the extended access program; average age 11.6 months). The same study involved five patients older than five years, three of whom were older than 10 years (10, 11 and 16 years) and received substitution therapy for more than 10 years.

All patients in the US trials and 19 out of 20 patients in the UK received enzyme replacement therapy during treatment, and all patients received preventive antimicrobials. A total of 19 out of 20 patients in the UK study and 29 out of 30 patients in the US studies received immunoglobulin replacement therapy; the remaining patients started immunoglobulin replacement therapy after infusion.

At the time of analysis, all patients underwent 24 months (USA) or 36 months (UK) of follow-up, with the exception of one patient from the USA and one patient from the UK, who were withdrawn from the study due to lack of sustained engraftment 5.9 months and 12.2 months after treatment, respectively. Both were subsequently treated with substitution therapy, and the first received donated blood stem cells.

Doctors did not detect monoclonal expansion, leukoproliferative complications, the appearance of a replicatively competent lentivirus or fatal outcomes in any of the patients. No autoimmune pathologies or cases of graft-versus-host disease were noted. Side effects, however, were reported in all patients: most of them were mild or moderate.

 During 24 months of follow-up in American studies, 73 out of 421 adverse events turned out to be severe and extremely severe, two of which (a decrease in the number of leukocytes and neutrophils) led to the withdrawal of one patient from the study: his transplanted modified blood stem cells took root, but did not multiply. Twelve patients had one or more serious adverse events. Most often these were infections (27 percent) and gastrointestinal diseases (17 percent), among which only one researchers associated with treatment.

During 36 months of follow-up in the UK study, 60 out of 321 adverse events were moderate to severe, and 25 were considered serious by doctors, and they occurred in 11 out of 20 patients. The most common of the serious adverse events was fever (in 30 percent of patients). One serious adverse event related to treatment turned out to be associated with known contamination of medicines.

In all studies, overall survival was 100 percent for 12 months and remained at 100 percent for 24 months for patients in all three studies; overall survival was still 100 percent for 36 months among patients in the UK study. After one year, the survival rate without adverse events was 97 percent among patients in the US study and 100 percent among patients in the UK study.

Thanks to the labeling of vector genes, scientists found out that three months after the introduction of the drug, genetically modified granulocytes and monocytes were detected in all patients, with the exception of one. In one patient in a British study, the concentration of genetically modified granulocytes and monocytes began to decrease after three months of follow-up. Also in a British study in patients, persistent gene labeling was observed in CD3+ T cells and CD19+ B cells, which increased over 24 months and remained stable up to 36 months.

In all three studies, the average activity of blood pressure in red blood cells increased sharply during the first three months and remained within or above the levels observed in healthy children. In studies in the USA, the median total level of deoxyadenosine nucleotides in erythrocytes remained well below the maximum threshold indicating adequate detoxification until the end of the studies. Similar results were observed in the British study.

Eventually, the number of lymphocytes in most patients reached the age norms for most populations of lymphocytes. In addition, the average number of naive T cells increased by about five times compared to the baseline level by the end of the studies in both countries, which indicates the restoration of thymopoiesis.

There was also an improvement in the function of B cells: in most patients, over time, the level of IgM and IgA in the blood serum increased to or above normal. In addition, 90 percent of Americans and 100 percent of Britons in the studies stopped immunoglobulin replacement therapy by 24 and 36 months, respectively.

At the same time, average IgG levels remained high after stopping immunoglobulin replacement therapy. Among patients who received tetanus vaccination, 4 out of 4 in America and 13 out of 14 in the UK had normal antibody reactions when tested at least three months after discontinuation of immunoglobulin replacement therapy.

In studies, the incidence of severe infections was generally low after gene therapy. Eight patients from the US have 15 serious infections, and seven patients in the UK have eight infections.

Thus, the steady engraftment of genetically modified cells led to the restoration of the activity of the ADA enzyme and the function of the immune system in 48 out of 50 children with severe combined immunodeficiency. Moreover, such gene therapy can be a reliable and viable treatment option for older children, for whom current treatment is not always effective and whose thymus function may already be reduced or absent altogether.

The success of gene therapy is forcing scientists and doctors around the world to increasingly turn to it in the treatment of seemingly incurable diseases. For example, Canadian scientists have already considered gene therapy for Fabry's disease safe, and with Leber's hereditary optical neuropathy, it improved vision in both eyes at once, although the drug was injected into only one.

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