Lentivector cryptic splicing mediates increase in CD34+ clones expressing truncated HMGA2 in human X-linked severe combined immunodeficiency.

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where 'emergency' life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken β-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety

Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu

\u3ci\u3eDrosophila\u3c/i\u3e Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu