Mutational spectrum of the succinate semialdehyde dehydrogenase (ALDH5A1) gene and functional analysis of 27 novel disease-causing mutations in patients with SSADH deficiency

Succinate semialdehyde dehydrogenase (SSADH; ALlDH5A1) deficiency, a rare metabolic disorder that disrupts the normal degradation of GABA, gives rise to a highly heterogeneous neurological phenotype ranging from mild to very severe. The nature of the mutation has so far been reported in patients from six families world wide and eight different mutations were described. Here we report the mutational spectrum in 48 additional unrelated families of different geographic origin. We detected 27 novel mutations at the cDNA level, of which 26 could be attributed to changes at the genomic level. Furthermore, six mutations were detected that did not strongly affect SSADH activity when expressed in HEK 293 cells and are considered nonpathogenic allelic variants. Twenty of the mutations were only found in one family. The spectrum of disease-causing mutations from all patients sequenced thus far consists of 25 point mutations, four small insertions, and five small deletions. Seven of these mutations affect splice junctions, seven are nonsense mutations, and 12 are missense mutations. Although there were no mutational hotspots or prevalent mutations responsible for a significant number of cases, 14 out of 37 (38%) of the missense alleles were present in exon 4 or 5. With one exception, the missense mutations we consider to be causative of SSADH deficiency reduced the SSADH activity to less than 5% of the normal activity in our in vitro expression system. This indicates that residual expression is not likely to be an important factor contributing to the large phenotypic differences observed among different families and even among siblings, suggesting that other modifying factors are of great importance in disease pathology. (C) 2003 Wiley,Liss, Inc

Characterization of AAV Integrations in preclinical models of gene therapy using RAAVioli pipeline with long and short sequencing reads

Adeno-Associated Viral vectors (AAVs) have been widely used in gene therapy to treat various genetic disorders. Although they are typically considered episomal vectors, sev- eral studies have shown that both fragmented and full-length AAV DNA can integrate into the genomes of host cells leading to hepatocellular carcinoma and clonal expansion in some pre- clinical models. However, methods and bioinformatic tools that provide a reliable and efficient assessment of AAV integration sites (IS) are required. Here, we propose a sonication-based PCR approach combined with short-read sequencing and a bioinformatics pipeline called RAAVIoli (Recombinant Adeno-Associated Viral Integration analysis) to characterize AAV integration sites (IS) and vector rearrangements. RAAVioli utilizes Python and R scripts to parse alignments, identify IS, and reconstruct vector rearrangements using CIGAR strings. The robustness of our approach was dem- onstrated by characterizing AAV IS in a humanized liver mouse model, where human primary hepatocytes were transduced with a tomato-expressing AAV. In this model, vector insertions were previously characterized using an AAV-specific probe base selection method and long-read PacBio sequencing. Sequencing reads were analyzed in both cases using the same RAAVioli pipeline. A greater number of AAV IS were identified by PCR/ short-read sequencing (N 1⁄4 730) as compared to the long-read sequence approach (N 1⁄4 370). AAV IS distributed similarly within the human genome showing the typical preference of tar- geting CpG islands and transcriptional start sites. Moreover, 32 IS were shared among the two datasets, demonstrating the consis- tency of the results obtained independently from the sequencing platform adopted. Although the short-read sequencing method identified a lower number of IS (*10%) with rearranged AAV genomes compared to the long-read sequencing approach (*25%), it demonstrates significantly greater efficiency in retrieving AAV insertion sites. Thanks to this platform, we recently characterized AAV IS in a liver-directed gene editing approach used to correct Wilson Disease (WD). WD is an autosomal recessive disorder caused by mutations in the ATP7B gene which is primarily expressed in hepatocytes and is involved in copper metabolism. Here, genome editing was accomplished by integrating a promoter- less human mini-ATP7B cDNA into the albumin locus (Alb- ATP7B) using a nuclease-free homology-directed repair (HDR) mechanism. The treatment corrected the disease pheno- type in Atp7b-/- mice by promoting liver repopulation of edited hepatocytes. Analysis of AAV IS revealed that vector insertion did not only cluster near or within the albumin-edited site but also distributed throughout the entire gene, exhibiting a strand-orientation bias. Indeed, 80% of the Albumin IS have the integrated vector genome oriented in the same direction as the targeted gene. Interestingly, this phenomenon was not observed in Atp7b-/- mice injected with a control vector expressing GFP. These data suggest the occurrence of a selec- tion mechanism favoring the survival of hepatocytes express- ing the therapeutic transgene consequently to integration events that are not driven by HDR-mediated mechanism. In summary, our work indicates that the development of reli- able methods for the characterization of AAV integrations is fun- damental to providing insights into the safety and efficacy of these vectors in several gene therapy applications

Hyperthermophilic Aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines

The placement of the extreme thermophile Aquifex aeolicus in the bacterial phylogenetic tree has evoked much controversy. We investigated whether adaptations for growth at high temperatures would alter a key functional component of the replication machinery, specifically DnaG primase. Although the structure of bacterial primases is conserved, the trinucleotide initiation specificity for A. aeolicus was hypothesized to differ from other microbes as an adaptation to a geothermal milieu. To determine the full range of A. aeolicus primase activity, two oligonucleotides were designed that comprised all potential trinucleotide initiation sequences. One of the screening templates supported primer synthesis and the lengths of the resulting primers were used to predict possible initiation trinucleotides. Use of trinucleotide-specific templates demonstrated that the preferred initiation trinucleotide sequence for A. aeolicus primase was 5′-d(CCC)-3′. Two other sequences, 5′-d(GCC)-3′ and d(CGC)-3′, were also capable of supporting initiation, but to a much lesser degree. None of these trinucleotides were known to be recognition sequences used by other microbial primases. These results suggest that the initiation specificity of A. aeolicus primase may represent an adaptation to a thermophilic environment

Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans

Background and Aims Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. Approach and Results Fah(-/-), Rag2(-/-), and Il2rg(-/-) knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. Conclusions LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.Peer reviewe

Nonvirally Modified Autologous Primary Hepatocytes Correct Diabetes and Prevent Target Organ Injury in a Large Preclinical Model

BACKGROUND: Current gene- and cell-based therapies have significant limitations which impede widespread clinical application. Taking diabetes mellitus as a paradigm, we have sought to overcome these limitations by ex vivo electrotransfer of a nonviral insulin expression vector into primary hepatocytes followed by immediate autologous reimplantation in a preclinical model of diabetes. METHODS AND RESULTS: In a single 3-hour procedure, hepatocytes were isolated from a surgically resected liver wedge, electroporated with an insulin expression plasmid ex vivo and reimplanted intraparenchymally under ultrasonic guidance into the liver in each of 10 streptozotocin-induced diabetic Yorkshire pigs. The vector was comprised of a bifunctional, glucose-responsive promoter linked to human insulin cDNA. Ambient glucose concentrations appropriately altered human insulin mRNA expression and C-peptide secretion within minutes in vitro and in vivo. Treated swine showed correction of hyperglycemia, glucose intolerance, dyslipidemia and other metabolic abnormalities for > or = 47 weeks. Metabolic correction correlated significantly with the number of hepatocytes implanted. Importantly, we observed no hypoglycemia even under fasting conditions. Direct intrahepatic implantation of hepatocytes did not alter biochemical indices of liver function or induce abnormal hepatic lobular architecture. About 70% of implanted hepatocytes functionally engrafted, appeared histologically normal, retained vector DNA and expressed human insulin for > or = 47 weeks. Based on structural tissue analyses and transcriptome data, we showed that early correction of diabetes attenuated and even prevented pathological changes in the eye, kidney, liver and aorta. CONCLUSIONS: We demonstrate that autologous hepatocytes can be efficiently, simply and safely modified by electroporation of a nonviral vector to express, process and secrete insulin durably. This strategy, which achieved significant and sustained therapeutic efficacy in a large preclinical model without adverse effects, warrants consideration for clinical development especially as it could have broader future applications for the treatment of other acquired and inherited diseases for which systemic reconstitution of a specific protein deficiency is critical

Mechanism of cell death resulting from DNA interstrand cross-linking in mammalian cells

DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G2) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death