The rapid development of CRISPR genome editing technology has provided the potential to treat genetic diseases effectively and precisely. However, efficient and safe delivery of genome editors to affected tissues remains a challenge. Here, we developed luminescent ABE (LumA), a luciferase reporter mouse model containing the R387X mutation (c.A1159T) in the luciferase gene located in the Rosa26 locus of the mouse genome. This mutation eliminates luciferase activity but can be restored upon A-to-G correction by SpCas9 adenine base editors (ABEs). The LumA mouse model was validated through intravenous injection of two FDA-approved lipid nanoparticle (LNP) formulations consisting of either MC3 or ALC-0315 ionizable cationic lipids, encapsulated with ABE mRNA and LucR387X-specific guide RNA (gRNA). Whole-body bioluminescence live imaging showed consistent restoration of luminescence lasting up to 4 months in treated mice. Compared with mice carrying the wild-type luciferase gene, the ALC-0315 and MC3 LNP groups showed 83.5% ± 17.5% and 8.4% ± 4.3% restoration of luciferase activity in the liver, respectively, as measured by tissue luciferase assays. These results demonstrated successful development of a luciferase reporter mouse model that can be used to evaluate the efficacy and safety of different genome editors, LNP formulations, and tissue-specific delivery systems for optimizing genome editing therapeutics.

Lipoprotein lipase deficiency (LPLD) results from mutations within the lipoprotein lipase (LPL) gene that lead to a complete lack of catalytically active LPL protein. Glybera was one of the first adeno-associated virus (AAV) gene replacement therapy to receive European Medicines Agency regulatory approval for the treatment of LPLD. However, Glybera is no longer marketed potentially due to a combination of economical, manufacturing, and vector-related issues. The aim of this study was to develop a more efficacious AAV gene therapy vector for LPLD. Following preclinical biodistribution, efficacy and non-Good Laboratory Practice toxicity studies with novel AAV1 and AAV8-based vectors in mice, we identified AAV8 pVR59. AAV8 pVR59 delivered a codon-optimized, human gain-of-function hLPLS447X transgene driven by a CAG promoter in an AAV8 capsid. AAV8 pVR59 was significantly more efficacious, at 10- to 100-fold lower doses, compared with an AAV1 vector based on Glybera, when delivered intramuscularly or intravenously, respectively, in mice with LPLD. Efficient gene transfer was observed within the injected skeletal muscle and liver following delivery of AAV8 pVR59, with long-term correction of LPLD phenotypes, including normalization of plasma triglycerides and lipid tolerance, for up to 6 months post-treatment. While intramuscular delivery of AAV8 pVR59 was well tolerated, intravenous administration augmented liver pathology. These results highlight the feasibility of developing a superior AAV vector for the treatment of LPLD and provide critical insight for initiating studies in larger animal models. The identification of an AAV gene therapy vector that is more efficacious at lower doses, when paired with recent advances in production and manufacturing technologies, will ultimately translate to increased safety and accessibility for patients.

Genome editing provides a new therapeutic strategy to cure genetic diseases. The recently developed CRISPR-Cas9 base editing technology has shown great potential to repair the majority of pathogenic point mutations in the patient's DNA precisely. Base editor is the fusion of a Cas9 nickase with a base-modifying enzyme that can change a nucleotide on a single strand of DNA without generating double-stranded DNA breaks. However, a major limitation in applying such a system is the prerequisite of a protospacer adjacent motif sequence at the desired position relative to the target site. Progress has been made to increase the targeting scope of base editors by engineering SpCas9 protein variants, establishing systems with broadened editing windows, characterizing new SpCas9 orthologs, and developing prime editing technology. In this review, we discuss recent progress in the development of CRISPR base editing, focusing on its targeting scope, and we provide a workflow for selecting a suitable base editor based on the target nucleotide sequences.

Recent advances in genome sequencing have greatly improved our ability to understand and identify the causes of genetic diseases. However, there remains an urgent need for innovative, safe, and effective treatments for these diseases. CRISPR-based genome editing systems have become important and powerful tools in the laboratory, and efforts are underway to translate these into patient therapies. Therapeutic base editing is one form of genome engineering that has gained much interest because of its simplicity, specificity, and effectiveness. Base editors are a fusion of a partially deactivated Cas9 enzyme with nickase function, together with a base-modifying enzyme. They are capable of precisely targeting and repairing a pathogenic mutation to restore the normal function of a gene, ideally without disturbing the rest of the genome. In the past year, research has identified new safety concerns of base editors and sparked new innovations to improve their safety. In this review, we provide an overview of the recent advances in the safety and effectiveness of therapeutic base editors and prime editing.

Clinical and laboratory data were collected from three Finnish patients including a sibling pair and another unrelated child with unexplained childhood hypoglycemia. Transient elevation of alanine transaminase, lactate and tricarboxylic acid cycle intermediates, especially fumarate, were noticed in urine organic acid analysis. Exome sequencing was performed for the patients and their parents. A novel homozygous PCK1 c.925G>A (p.G309R) mutation was detected in all affected individuals. COS-1 cells transfected with mutant PCK1 transcripts were used to study the pathogenic nature of the detected variant. The COS-1 transfected cells showed the mutant gene to be incapable of producing a normally functioning cytosolic phosphoenolpyruvate carboxykinase (PEPCK) enzyme. This report further delineates the clinical phenotype of isolated cytosolic PEPCK deficiency and offers a metabolic pattern helping to recognize these patients. Cytosolic PEPCK deficiency should be considered in the differential diagnosis of children presenting with hypoglycemia, hepatic dysfunction and elevated tricarboxylic acid intermediates in urinary organic acid analysis.

Objective— High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate–binding cassette transporter A8 ( ABCA8 ) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. Approach and Results— We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P =0.005). HDLc levels were significantly decreased by 29% ( P =0.01) in Abca8b −/− mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P =0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate–binding cassette transporter A1 and further potentiates adenosine triphosphate–binding cassette transporter A1–mediated cholesterol efflux. Conclusions— ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.

BACKGROUND:Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level.METHODS:To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes.RESULTS:We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%).CONCLUSIONS:Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

Primary 5-oxoprolinuria (pyroglutamic aciduria) is caused by a genetic defect in the γ-glutamyl cycle, affecting either glutathione synthetase or 5-oxoprolinase. While several dozens of patients with glutathione synthetase deficiency have been reported, with hemolytic anemia representing the clinical key feature, 5-oxoprolinase deficiency due to OPLAH mutations is less frequent and so far has not attracted much attention. This has prompted us to investigate the clinical phenotype as well as the underlying genotype in patients from 14 families of various ethnic backgrounds who underwent diagnostic mutation analysis following the detection of 5-oxoprolinuria. In all patients with 5-oxoprolinuria studied, bi-allelic mutations in OPLAH were indicated. An autosomal recessive mode of inheritance for 5-oxoprolinase deficiency is further supported by the identification of a single mutation in all 9/14 parent sample sets investigated (except for the father of one patient whose result suggests homozygosity), and the absence of 5-oxoprolinuria in all tested heterozygotes. It is remarkable, that all 20 mutations identified were novel and private to the respective families. Clinical features were highly variable and in several sib pairs, did not segregate with 5-oxoprolinuria. Although a pathogenic role of 5-oxoprolinase deficiency remains possible, this is not supported by our findings. Additional patient ascertainment and long-term follow-up is needed to establish the benign nature of this inborn error of metabolism. It is important that all symptomatic patients with persistently elevated levels of 5-oxoproline and no obvious explanation are investigated for the genetic etiology.

We report a patient from a consanguineous family who presented with transient acute liver failure and biochemical patterns suggestive of disturbed urea cycle and mitochondrial function, for whom conventional genetic and metabolic investigations for acute liver failure failed to yield a diagnosis. Whole exome sequencing revealed a homozygous 12-bp deletion in PCK1 (MIM 614168) encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK); enzymatic studies subsequently confirmed its pathogenic nature. We propose that PEPCK deficiency should be considered in the young child with unexplained liver failure, especially where there are marked, accumulations of TCA cycle metabolites on urine organic acid analysis and/or an amino acid profile with hyperammonaemia suggestive of a proximal urea cycle defect during the acute episode. If suspected, intravenous administration of dextrose should be initiated. Long-term management comprising avoidance of fasting with the provision of a glucose polymer emergency regimen for illness management may be sufficient to prevent future episodes of liver failure. This case report provides further insights into the (patho-)physiology of energy metabolism, confirming the power of genomic analysis of unexplained biochemical phenotypes.

We describe neurotransmitter abnormalities in two patients with drug-resistant epilepsy resulting from deleterious de novo mutations in sodium channel genes. Whole exome sequencing identified a de novo SCN2A splice-site mutation (c.2379+1G>A, p.Glu717Gly.fs*30) resulting in deletion of exon 14, in a 10-year old male with early onset global developmental delay, intermittent ataxia, autism, hypotonia, epileptic encephalopathy and cerebral/cerebellar atrophy. In the cerebrospinal fluid both homovanillic acid and 5-hydroxyindoleacetic acid were significantly decreased; extensive biochemical and genetic investigations ruled out primary neurotransmitter deficiencies and other known inborn errors of metabolism. In an 8-year old female with an early onset intractable epileptic encephalopathy, developmental regression, and progressive cerebellar atrophy, a previously unreported de novo missense mutation was identified in SCN8A (c.5615G>A; p.Arg1872Gln), affecting a highly conserved residue located in the C-terminal of the Nav1.6 protein. Aside from decreased homovanillic acid and 5-hydroxyindoleacetic acid, 5-methyltetrahydrofolate was also found to be low. We hypothesize that these channelopathies cause abnormal synaptic mono-amine metabolite secretion/uptake via impaired vesicular release and imbalance in electrochemical ion gradients, which in turn aggravate the seizures. Treatment with oral 5-hydroxytryptophan, l-Dopa/Carbidopa, and a dopa agonist resulted in mild improvement of seizure control in the male case, most likely via dopamine and serotonin receptor activated signal transduction and modulation of glutamatergic, GABA-ergic and glycinergic neurotransmission. Neurotransmitter analysis in other sodium channelopathy patients will help validate our findings, potentially yielding novel treatment opportunities.

Introduction Mutations in the family of SCN genes encoding sodium channels are responsible for several disorders affecting the central and peripheral nervous systems and muscle. Disease arising from sodium channel mutants range from the relatively benign (e.g. mild myotonia) to the fatal (e.g. long-QT syndrome), with a wide variety of disorders spanning the spectrum of severity. Identified SCN4a mutations to date have been consistently autosomal dominant and associated with paramyotonia congenita, potassium-mediated periodic paralysis or aggravated myotonia due to defects altering the biophysical properties of sodium channels that mediate membrane hyper- or hypo-excitability. Here we describe a newly recognised autosomal-recessive syndrome comprising severe congenital hypotonia with respiratory failure in a family of Punjabi descent, with 2 of 3 children affected. Methods and results Using whole exome sequencing we identified two new mutations (g. 62025363 C >T, D1069N and g. 62025425 T >G, splice site) in the SCN4A gene, confirmed via Sanger sequencing. Reverse transcriptase polymerase chain reaction shows that the splice-site mutation in SCN4A leads to altered RNA. To investigate the impact of the missense mutation, c.3205G >A, Chinese hamster ovary (CHOk1) cells transfected with either a WT or D1069N SCN4A were examined for their biophysical properties. A set of depolarizing test pulses was used to measure the voltage dependence of activation and indicated biophysical changes in the encoded voltage-gated sodium channel (NaV1.4). Conclusions Together, our findings characterise the first reported evidence of an autosomal recessive SCN4a sodium channelopathy comprising severe congenital neuromuscular hypotonia and respiratory failure with biophysical dysfunction of NaV1.4 attributable to SCN4a compound heterozygous gene mutation.

Congenital myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by congenital or early-onset hypotonia and muscle weakness, and specific pathological features on muscle biopsy. The phenotype ranges from foetal akinesia resulting in in utero or neonatal mortality, to milder disorders that are not life-limiting. Over the past decade, more than 20 new congenital myopathy genes have been identified. Most encode proteins involved in muscle contraction; however, mutations in ion channel-encoding genes are increasingly being recognized as a cause of this group of disorders. SCN4A encodes the α-subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4). This channel is essential for the generation and propagation of the muscle action potential crucial to muscle contraction. Dominant SCN4A gain-of-function mutations are a well-established cause of myotonia and periodic paralysis. Using whole exome sequencing, we identified homozygous or compound heterozygous SCN4A mutations in a cohort of 11 individuals from six unrelated kindreds with congenital myopathy. Affected members developed in utero- or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth. The remaining four cases had marked congenital or neonatal-onset hypotonia and weakness associated with mild-to-moderate facial and neck weakness, significant neonatal-onset respiratory and swallowing difficulties and childhood-onset spinal deformities. All four surviving cohort members experienced clinical improvement in the first decade of life. Muscle biopsies showed myopathic features including fibre size variability, presence of fibrofatty tissue of varying severity, without specific structural abnormalities. Electrophysiology suggested a myopathic process, without myotonia. In vitro functional assessment in HEK293 cells of the impact of the identified SCN4A mutations showed loss-of-function of the mutant Nav1.4 channels. All, apart from one, of the mutations either caused fully non-functional channels, or resulted in a reduced channel activity. Each of the affected cases carried at least one full loss-of-function mutation. In five out of six families, a second loss-of-function mutation was present on the trans allele. These functional results provide convincing evidence for the pathogenicity of the identified mutations and suggest that different degrees of loss-of-function in mutant Nav1.4 channels are associated with attenuation of the skeletal muscle action potential amplitude to a level insufficient to support normal muscle function. The results demonstrate that recessive loss-of-function SCN4A mutations should be considered in patients with a congenital myopathy.

The causality of the inverse correlation between cardiovascular disease risk and high density lipoprotein cholesterol (HDLc) levels is unclear, raising the need for further studies. We identified and characterized the impact of a novel HDLc gene, the ATP binding cassette transporter A8 (ABCA8). We sequenced ABCA8 in 80 low HDLc (HDLc %ile≤10th) and 120 high HDLc (HDLc %ile≥90th) individuals, and identified three ABCA8 variants exclusively in low-HDLc subjects: Proline609Arginine (in the ATP-binding domain), E17-2 A>G (disruption of essential splice site) and Threonine741Stop.

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.

Tangier disease is a rare, autosomal recessive disorder caused by mutations in the ABCA1 gene and is characterized by near absence of plasma high-density lipoprotein cholesterol, accumulation of cholesterol in multiple tissues, peripheral neuropathy, and accelerated atherosclerosis. Here we report three new kindreds with Tangier disease harboring both known and novel mutations in ABCA1. One patient was identified to be homozygous for a nonsense mutation, p.Gln1038*. In a remarkably large Tangier disease pedigree with four affected siblings, we identified compound heterozygosity for previously reported missense variants, p.Arg937Val and p.Thr940Met, and show that both of these mutations result in significantly impaired cholesterol efflux in transfected cells. In a third pedigree, the proband was identified to be compound heterozygous for two novel mutations, a frameshift (p.Ile1200Hisfs*4) and an intronic variant (c.4176-11T>G), that lead to the creation of a cryptic splice site acceptor and premature truncation, p.Ser1392Argfs*6. We demonstrate that this mutation arose de novo, the first demonstration of a pathogenic de novo mutation in ABCA1 associated with Tangier disease. We also report results of glucose tolerance testing in a Tangier disease kindred for the first time, showing a gene-dose relationship between ABCA1 activity and glucose tolerance and suggesting that Tangier disease patients may have substantially impaired islet function. Our findings provide insight into the diverse phenotypic manifestations of this rare disorder, expand the list of pathogenic mutations in ABCA1, and increase our understanding of how specific mutations in this gene lead to abnormal cellular and physiological phenotypes.

HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β -chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β -chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β -chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β -chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β -chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β -chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

Objective—The ATP-binding cassette transporter A1 (ABCA1) protein maintains cellular cholesterol homeostasis in several different tissues. In the liver, ABCA1 is crucial for high-density lipoprotein biogenesis, and in the pancreas ABCA1 can regulate insulin secretion. In this study, our aim was to identify novel microRNAs that regulate ABCA1 expression in these tissues. Approach and Results—We combined multiple microRNA prediction programs to identify 8 microRNAs that potentially regulate ABCA1. A luciferase reporter assay demonstrated that 5 of these microRNAs (miR-148, miR-27, miR-144, miR-145, and miR-33a/33b) significantly repressed ABCA1 3′-untranslated region activity with miR-145 resulting in one of the larger decreases. In hepatic HepG2 cells, miR-145 can regulate both ABCA1 protein expression levels and cholesterol efflux function. In murine islets, an increase in miR-145 expression decreased ABCA1 protein expression, increased total islet cholesterol levels, and decreased glucose-stimulated insulin secretion. Inhibiting miR-145 produced the opposite effect of increasing ABCA1 protein levels and improving glucose-stimulated insulin secretion. Finally, increased glucose levels in media significantly decreased miR-145 levels in cultured pancreatic beta cells. These findings suggest that miR-145 is involved in glucose homeostasis and is regulated by glucose concentration. Conclusions—Our studies demonstrate that miR-145 regulates ABCA1 expression and function, and inhibiting this microRNA represents a novel strategy for increasing ABCA1 expression, promoting high-density lipoprotein biogenesis in the liver, and improving glucose-stimulated insulin secretion in islets.