A Comprehensive Study of the Effects by Sequence Truncation within Inverted Terminal Repeats (ITRs) on the Productivity, Genome Packaging, and Potency of AAV Vectors.

One of the primary challenges in working with adeno-associated virus (AAV) lies in the inherent instability of its inverted terminal repeats (ITRs), which play vital roles in AAV replication, encapsidation, and genome integration. ITRs contain a high GC content and palindromic structure, which occasionally results in truncations and mutations during plasmid amplification in bacterial cells. However, there is no thorough study on how these alterations in ITRs impact the ultimate AAV vector characteristics. To close this gap, we designed ITRs with common variations, including a single B, C, or D region deletion at one end, and dual deletions at both ends of the vector genome. These engineered ITR-carrying plasmids were utilized to generate AAV vectors in HEK293 cells. The crude and purified AAV samples were collected and analyzed for yield, capsid DNA-filled percentage, potency, and ITR integrity. The results show that a single deletion had minor impact on AAV productivity, packaging efficiency, and in vivo potency. However, deletions on both ends, except A, showed significant negative effects on the above characteristics. Our work revealed the role of ITR regions, A, B, C, and D for AAV production and DNA replication, and proposes a new strategy for the quality control of ITR-bearing plasmids and final AAV products.

Novel AAV-mediated genome editing therapy improves health and survival in a mouse model of methylmalonic acidemia.

Methylmalonic acidemia (MMA) is an inborn error of metabolism mostly caused by mutations in the mitochondrial methylmalonyl-CoA mutase gene (MMUT). MMA patients suffer from frequent episodes of metabolic decompensation, which can be life threatening. To mimic both the dietary restrictions and metabolic decompensation seen in MMA patients, we developed a novel protein-controlled diet regimen in a Mmut deficient mouse model of MMA and demonstrated the therapeutic benefit of mLB-001, a nuclease-free, promoterless recombinant AAV GeneRideTM vector designed to insert the mouse Mmut into the endogenous albumin locus via homologous recombination. A single intravenous administration of mLB-001 to neonatal or adult MMA mice prevented body weight loss and mortality when challenged with a high protein diet. The edited hepatocytes expressed functional MMUT protein and expanded over time in the Mmut deficient mice, suggesting a selective growth advantage over the diseased cells. In mice with a humanized liver, treatment with a human homolog of mLB-001 resulted in site-specific genome editing and transgene expression in the transplanted human hepatocytes. Taken together, these findings support the development of hLB-001 that is currently in clinical trials in pediatric patients with severe forms of MMA.

Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation.

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers.

Stress-related hormonal suppression of natural killer activity does not show menstrual cycle variations: implications for timing of surgery for breast cancer.

BACKGROUND: Evidence exists that prognosis in breast cancer may be related to the timing of primary surgery. Fluctuations in natural killer (NK) cells and/or their sensitivity to surgical stress hormones could mediate this phenomenon. We sought to establish, firstly, if there are any numerical or functional changes in NK cells during the course of the menstrual cycle and, secondly, if their prior in vitro incubation with cortisol and adrenaline produced functional change that was more pronounced at a particular point in the cycle. SUBJECTS AND METHODS: Normally menstruating females (n = 10) were sampled at two points (mid-follicular and mid-luteal) during two cycles. NK cell numbers were determined by flow cytometry. Natural killer cell activity (NKA) (unstimulated and following a 2-hour pre-incubation with cortisol and adrenaline) was determined using chromium-51 release assays from peripheral blood mononuclear cells (PBMCs) prepared from cryopreserved samples from the second cycle. RESULTS: NK cell numbers (expressed as a percentage of a standard lymphgate) were significantly higher at the mid-luteal point compared to the mid-follicular point (8.45 +/- 3.71 vs. 6.85 +/- 2.80, p < 0.05, data pooled from both cycles). Corresponding changes in NKA were observed, although these did not reach statistical significance. The mean inhibitory effects of cortisol (67%) and adrenaline (12%) on NKA were significant, but this phenomenon was uninfluenced by cycle point. CONCLUSION: This study supports the role of menstrual cycle-induced alterations in NK cells as a putative mediator of improved survival when surgery for breast cancer is carried out in the follicular phase. However there is no evidence of the cyclical sensitivity of NK cells to the principal surgical stress hormones.

The effect of lossy ECG compression on QRS and HRV feature extraction.

This paper describes the performance of beat detection and heart rate variability (HRV) feature extraction on electrocardiogram signals which have been compressed and reconstructed with a lossy compression algorithm. The set partitioning in hierarchical trees (SPIHT) compression algorithm was used with sixteen compression ratios (CR) between 2 and 50 over the records of the MIT/BIH arrhythmia database. Sensitivities and specificities between 99% and 85% were computed for each CR utilised. The extracted HRV features were between 99% and 82% similar to the features extracted from the annotated records. A notable accuracy drop over all features extracted was noted beyond a CR of 30, with falls of 10% accuracy beyond this compression ratio.

Cortisol does not mediate the suppressive effects of psychiatric morbidity on natural killer cell activity: a cross-sectional study of patients with early breast cancer.

BACKGROUND: There is evidence that depression impairs natural killer cell activity (NKA); this could have implications for anti-tumour immunity. Our aim was to examine the role of the hypothalamic-pituitary-adrenal (HPA) axis in suppressing NKA in a population of patients with early breast cancer, screened for depression. Secondary aims were to study the relationship between psychological, endocrine and immune variables and baseline tumour characteristics. METHOD: A cross-sectional population of female patients (n=55) with early breast cancer was sampled prior to primary surgery. Structured interview and psychometric instruments measured psychological distress. Flow cytometry was used to enumerate NK cells and lymphocytes were cryopreserved for use in a 51Cr-release assay, to estimate NKA. Midnight and three early morning saliva samples were collected to measure free cortisol levels. Tumour characteristics were obtained from hospital laboratory data. RESULTS: A high rate of psychological morbidity (40%) was observed in the population. NKA was reduced in those with past or current psychiatric illness compared to those without (344 v. 553 LU20 and 455 v. 569 LU20 respectively, p < 0.05 for both). Cortisol was not related to psychological status but was modestly positively correlated to NKA. A positive correlation was observed between the Fighting Spirit subscale of the Mental Adjustment to Cancer Scale and tumour size (r=0.383, p=0.012). CONCLUSIONS: Our data support the evidence that psychological morbidity is associated with immune dysfunction; however, the most obvious candidate mediator of this effect, the HPA axis, does not appear responsible for this effect. Possible reasons for this are discussed.