Auto-expansion of in vivo HDAd-transduced hematopoietic stem cells by constitutive expression of tHMGA2.

We developed an in vivo hematopoietic stem cell (HSC) gene therapy approach that does not require cell transplantation. To achieve therapeutically relevant numbers of corrected cells, we constructed HSC-tropic HDAd5/35++ vectors expressing a 3' UTR truncated HMGA2 gene and a GFP reporter gene. A SB100x transposase vector mediated random integration of the tHMGA2/GFP transgene cassette. HSCs in mice were mobilized by subcutaneous injections of G-CSF and AMD3100/Plerixafor and intravenously injected with the integrating tHMGA2/GFP vector. This resulted in a slow but progressive, competitive expansion of GFP+ PBMCs, reaching about 50% by week 44 with further expansion in secondary recipients. Expansion occurred at the level of HSCs as well as at the levels of myeloid, lymphoid, and erythroid progenitors within the bone marrow and spleen. Importantly, based on genome-wide integration site analyses, expansion was polyclonal, without any signs of clonal dominance. Whole-exome sequencing did not show significant differences in the genomic instability indices between tHGMGA2/GFP mice and untreated control mice. Auto-expansion by tHMGA2 was validated in humanized mice. This is the first demonstration that simple injections of mobilization drugs and HDAd vectors can trigger auto-expansion of in vivo transduced HSCs resulting in transgene-marking rates that, theoretically, are curative for hemoglobinopathies.

Structural and genetic diversity in the secreted mucins MUC5AC and MUC5B.

The secreted mucins MUC5AC and MUC5B are large glycoproteins that play critical defensive roles in pathogen entrapment and mucociliary clearance. Their respective genes contain polymorphic and degenerate protein-coding variable number tandem repeats (VNTRs) that make the loci difficult to investigate with short reads. We characterize the structural diversity of MUC5AC and MUC5B by long-read sequencing and assembly of 206 human and 20 nonhuman primate (NHP) haplotypes. We find that human MUC5B is largely invariant (5,761-5,762 amino acids [aa]); however, seven haplotypes have expanded VNTRs (6,291-7,019 aa). In contrast, 30 allelic variants of MUC5AC encode 16 distinct proteins (5,249-6,325 aa) with cysteine-rich domain and VNTR copy-number variation. We group MUC5AC alleles into three phylogenetic clades: H1 (46%, ∼5,654 aa), H2 (33%, ∼5,742 aa), and H3 (7%, ∼6,325 aa). The two most common human MUC5AC variants are smaller than NHP gene models, suggesting a reduction in protein length during recent human evolution. Linkage disequilibrium and Tajima's D analyses reveal that East Asians carry exceptionally large blocks with an excess of rare variation (p < 0.05) at MUC5AC. To validate this result, we use Locityper for genotyping MUC5AC haplogroups in 2,600 unrelated samples from the 1000 Genomes Project. We observe a signature of positive selection in H1 among East Asians and a depletion of the likely ancestral haplogroup (H3). In Europeans, H3 alleles show an excess of common variation and deviate from Hardy-Weinberg equilibrium (p < 0.05), consistent with heterozygote advantage and balancing selection. This study provides a generalizable strategy to characterize complex protein-coding VNTRs for improved disease associations.

Structural and genetic diversity in the secreted mucins, MUC5AC and MUC5B.

The secreted mucins MUC5AC and MUC5B play critical defensive roles in airway pathogen entrapment and mucociliary clearance by encoding large glycoproteins with variable number tandem repeats (VNTRs). These polymorphic and degenerate protein coding VNTRs make the loci difficult to investigate with short reads. We characterize the structural diversity of MUC5AC and MUC5B by long-read sequencing and assembly of 206 human and 20 nonhuman primate (NHP) haplotypes. We find that human MUC5B is largely invariant (5761-5762aa); however, seven haplotypes have expanded VNTRs (6291-7019aa). In contrast, 30 allelic variants of MUC5AC encode 16 distinct proteins (5249-6325aa) with cysteine-rich domain and VNTR copy number variation. We grouped MUC5AC alleles into three phylogenetic clades: H1 (46%, ~5654aa), H2 (33%, ~5742aa), and H3 (7%, ~6325aa). The two most common human MUC5AC variants are smaller than NHP gene models, suggesting a reduction in protein length during recent human evolution. Linkage disequilibrium (LD) and Tajima's D analyses reveal that East Asians carry exceptionally large MUC5AC LD blocks with an excess of rare variation (p<0.05). To validate this result, we used Locityper for genotyping MUC5AC haplogroups in 2,600 unrelated samples from the 1000 Genomes Project. We observed signatures of positive selection in H1 and H2 among East Asians and a depletion of the likely ancestral haplogroup (H3). In Africans and Europeans, H3 alleles show an excess of common variation and deviate from Hardy-Weinberg equilibrium, consistent with heterozygote advantage and balancing selection. This study provides a generalizable strategy to characterize complex protein coding VNTRs for improved disease associations.

In vivo base editing by a single i.v. vector injection for treatment of hemoglobinopathies.

Individuals with ß-thalassemia or sickle cell disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% fetal hemoglobin (HbF) appear to be symptom free. Here, we used a nonintegrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113 A>G HPFH mutation in the γ-globin promoters in healthy CD46/ß-YAC mice carrying the human ß-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only s.c. and i.v. injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/ß-YAC mice resulted in > 60% -113 A>G conversion, with 30% γ-globin of ß-globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-Seq. In vitro, in HSCs from ß-thalassemia and patients with sickle cell disease, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.