The concept of natural genome reconstruction.Part 6. Analysis of changes in the frequency of occurrence of SNPs in exons of the genes associated with clonal hematopoiesis of uncertain potential in a patient with a neuroendocrine tumor of the small intestine in the terminal stage of progression after modification of the hematopoietic stem cell genome with hDNAgr (a clinical case report).

The final part of our series of studies reports the results of a pilot study of a clinical case (patient K.), indicating that therapy enabling reconstruction of the genome of hematopoietic stem cells (HSCs) reduces the frequency of SNPs in exons of the genes associated with clonal hematopoiesis of uncertain potential in a patient with a neuroendocrine tumor of the small intestine in the terminal stage of progression. The fundamental principles of the therapy were as follows. Control samples of peripheral blood mononuclear cells (baseline) were collected from the patient. CD34+ HSCs were then mobilized and collected. The HSC genome was modified using a technology enabling ex vivo correction of the nucleotide sequences of DNA chromosomes of poorly differentiated hematopoietic progenitor cells. For this purpose, the collected leukocyte suspension enriched with CD34+ HSCs was treated with fragmented deproteinized genomic DNA obtained from approximately 100 young healthy women in labor (hDNAgr). The patient was reinfused intravenously with the treated cells. Five days before reinfusion, the patient received immunosuppressive therapy. Samples of peripheral blood mononuclear cells were also collected 4, 8, 12, and 27 months after the therapeutic intervention. Full-exome sequencing of DNA isolated from selected cell samples was performed. A group of commonly accepted clonal hematopoiesis genes was selected as a criterion demonstrating changes in the genome. The following multiplex panels were used at the initial time point as well as time points after 4, 8, and 12 months: MGIEasy Exome Capture V5 Probe set, Roche KAPA HyperExome, Nanodigmbio NEXome Plus Panel v1.0, covering the entire exome and non-coding regions of the analyzed genes adjacent to exons with ~100× coverage. A ~1000× coated panel (Nanodigmbio NanOnco Plus Panel v3.0), including the genes with mutant alleles identified at the initial time point, was used to verify the identified mutations and more accurately determine their proportions in readings at the last stage of the analysis (27 months after the therapy). Whole-exome sequencing of hDNAgr (Roche KAPA HyperExome), which is used to reconstruct the genome of HSCs whose fragments collectively constitute the complete human genome, was also performed. Although the analysis of readings of whole-exome sequencing of DNA isolated from blood samples after treatment did not reveal any currently known signs of clonal hematopoiesis with uncertain potential in the patient K., we found a significant, within the framework of the selected criterion, decrease in the frequency range of SNPs to 18 % by 27 months of follow-up in 15 % cases of the heterozygous alleles (DNMT1, SF3B1(1-2)). Subsequent analysis of the frequency of occurrence of controlled SNPs and the corresponding data on the depth of their sequencing, performed using the All-FIT algorithm, indicates that by the 27th month of follow-up, the proportion of cells carrying the basic set of SNPs significantly decreases by 12 % (confidence interval (CI) = 5-16 %). In other words, in a mixture of cells heterozygous for this SNP, a population of cells carrying a homozygote without an SNP appears. Our findings may indicate that a significant proportion of cells underwent correction of the SNP to an alternative allele, and this correction was associated with the treatment of the original HSCs with hDNAgr. The detected changes in the group of clonal hematopoiesis genes suggest the possibility of correcting other loci throughout the HSCs genome. Therefore, the clinical manifestations in the development of the disease show a pronounced positive trend, which has persisted for four years, until present. It is assumed that the positive clinical outcome after therapy with reconstructed hematopoietic stem cells is associated with an increase in the regenerative potential of HSCs, which resulted from the genetic correction of unfavorable mutations in the HSCs genome.