Genetic therapies for neurological disorders

In recent years, it has become increasingly apparent that many neurological disorders are underpinned by a genetic aetiology. This has resulted in considerable efforts to develop therapeutic strategies which can treat the disease-causing mutation, either by supplying a functional copy of the mutated gene or editing the genomic sequence. In this review, we will discuss the main genetic strategies which are currently being explored for the treatment of monogenic neurological disorders, as well as some of the challenges they face. In addition, we will address some of the ethical difficulties which may arise.

Mice Harboring a Non-Functional CILK1/ICK Allele Fail to Model the Epileptic Phenotype in Patients Carrying Variant CILK1/ICK

CILK1 (ciliogenesis associated kinase 1)/ICK (intestinal cell kinase) is a highly conserved protein kinase that regulates primary cilia structure and function. CILK1 mutations cause a wide spectrum of human diseases collectively called ciliopathies. While several CILK1 heterozygous variants have been recently linked to juvenile myoclonic epilepsy (JME), it remains unclear whether these mutations cause seizures. Herein, we investigated whether mice harboring either a heterozygous null Cilk1 (Cilk1+/−) mutation or a heterozygous loss-of-function Cilk1 mutation (Cilk1R272Q/+) have epilepsy. We first evaluated the spontaneous seizure phenotype of Cilk1+/− and Cilk1R272Q/+ mice relative to wildtype littermates. We observed no electrographic differences among the three mouse genotypes during prolonged recordings. We also evaluated electrographic and behavioral responses of mice recovering from isoflurane anesthesia, an approach recently used to measure seizure-like activity. Again, we observed no electrographic or behavioral differences in control versus Cilk1+/− and Cilk1R272Q/+ mice upon isoflurane recovery. These results indicate that mice bearing a non-functional copy of Cilk1 fail to produce electrographic patterns resembling those of JME patients with a variant CILK1 copy. Our findings argue against CILK1 haploinsufficiency being the mechanism that links CILK1 variants to JME.

The Genetic Basis of Differential Autodiploidization in Evolving Yeast Populations

Spontaneous whole-genome duplication, or autodiploidization, is a common route to adaptation in experimental evolution of haploid budding yeast populations. The rate at which autodiploids fix in these populations appears to vary across strain backgrounds, but the genetic basis of these differences remains poorly characterized. Here we show that the frequency of autodiploidization differs dramatically between two closely related laboratory strains of Saccharomyces cerevisiae, BY4741 and W303. To investigate the genetic basis of this difference, we crossed these strains to generate hundreds of unique F1 segregants and tested the tendency of each segregant to autodiplodize across hundreds of generations of laboratory evolution. We find that variants in the SSD1 gene are the primary genetic determinant of differences in autodiploidization. We then used multiple laboratory and wild strains of S. cerevisiae to show that clonal populations of strains with a functional copy of SSD1 autodiploidize more frequently in evolution experiments, while knocking out this gene or replacing it with the W303 allele reduces autodiploidization propensity across all genetic backgrounds tested. These results suggest a potential strategy for modifying rates of spontaneous whole-genome duplications in laboratory evolution experiments in haploid budding yeast. They may also have relevance to other settings in which eukaryotic genome stability plays an important role, such as biomanufacturing and the treatment of pathogenic fungal diseases and cancers.

The Genetic Basis of Differential Autodiploidization in Evolving Yeast Populations

ABSTRACTSpontaneous whole-genome duplication, or autodiploidization, is a common route to adaptation in experimental evolution of haploid budding yeast populations. The rate at which autodiploids fix in these populations appears to vary across strain backgrounds, but the genetic basis of these differences remains poorly characterized. Here we show that the frequency of autodiploidization differs dramatically between two closely related laboratory strains ofSaccharomyces cerevisiae, BY4741 and W303. To investigate the genetic basis of this difference, we crossed these strains to generate hundreds of unique F1 segregants and tested the tendency of each segregant to autodiplodize across hundreds of generations of laboratory evolution. We find that variants in theSSD1gene are the primary genetic determinant of differences in autodiploidization. We then used multiple laboratory and wild strains ofS. cerevisiaeto show that clonal populations of strains with a functional copy ofSSD1autodiploidize more frequently in evolution experiments, while knocking out this gene or replacing it with the W303 allele reduces autodiploidization propensity across all genetic backgrounds tested. These results suggest a potential strategy for modifying rates of spontaneous whole-genome duplications in laboratory evolution experiments in haploid budding yeast. They may also have relevance to other settings in which eukaryotic genome stability plays an important role, such as biomanufacturing and the treatment of pathogenic fungal diseases and cancers.

OR25-01 Durable CYP21A2 Gene Therapy in Non-Human Primates for Treatment of Congenital Adrenal Hyperplasia

Severe Congenital Adrenal Hyperplasia (CAH) is most commonly caused by genetic defects in the CYP21A2 gene, which leads to a deficiency of 21-hydroxylase enzyme and disruption in the biosynthesis of Adrenal corticosteriods. Despite treatment with corticosteroids, patients remain at significant risk for adrenal crisis, experiencing a 3-fold higher mortality rate than age matched controls. They also suffer from significant infertility, bone, metabolic, and cardiovascular disease, and hyperandrogenism in women leading to genital abnormalities, hirsutism, and other complications. We are developing an AAV5- based gene therapy (BBP-631) that will provide a functional copy of the CYP21A2 gene to the adrenal glands of CAH patients. To determine the durability of this therapy we treated cynomolgus monkeys with increasing doses of BBP-631 via intravenous injection. At 4-, 12- and 24-weeks post treatment, expression of hCYP21A2 mRNA and vector genome copies (VGC) in the adrenals and other peripheral tissues was measured. VGC was present in the liver and adrenals at 4 weeks, with durable detection through 24 weeks and total vg levels were dose dependent. hCYP21A2 RNA expression in adrenal and liver tissues was also dose dependent and continued to increase from 4 weeks through 12 weeks. There were no adverse safety signals in any of the treated animals. This data combined with efficacy data of BBP-631 in a Cyp21-/- mouse model supports our continued clinical development of BBP-631 as a treatment for congenital adrenal hyperplasia.

An osmolality/ salinity-responsive enhancer 1 (OSRE1) in intron 1 promotes salinity induction of tilapia glutamine synthetase

ABSTRACTEuryhaline tilapia (Oreochromis mossambicus) are fish that tolerate a wide salinity range from fresh water to >3x seawater. Even though the physiological effector mechanisms of osmoregulation that maintain plasma homeostasis in fresh water and seawater fish are well known, the corresponding molecular mechanisms that control switching between hyper- (fresh water) and hypo-osmoregulation (seawater) remain mostly elusive. In this study we show that hyperosmotic induction of glutamine synthetase represents a prominent part of this switch. Proteomics analysis of the O. mossambicus OmB cell line revealed that glutamine synthetase is transcriptionally regulated by hyperosmolality. Therefore, the 5’ regulatory sequence of O. mossambicus glutamine synthetase was investigated. Using an enhancer trapping assay, we discovered a novel osmosensitive mechanism by which intron 1 positively mediates glutamine synthetase transcription. Intron 1 includes a single, functional copy of an osmoresponsive element, osmolality/salinity-responsive enhancer 1 (OSRE1). Unlike for conventional enhancers, the hyperosmotic induction of glutamine synthetase by intron 1 is position dependent. But irrespective of intron 1 position, OSRE1 deletion from intron 1 abolishes hyperosmotic enhancer activity. These findings indicate that proper intron 1 positioning and the presence of an OSRE1 in intron 1 are required for precise enhancement of hyperosmotic glutamine synthetase expression.

Systemic AAV8-mediated delivery of a functional copy of muscle glycogen phosphorylase (Pygm) ameliorates disease in a murine model of McArdle disease

McArdle disease is a disorder of carbohydrate metabolism that causes painful skeletal muscle cramps and skeletal muscle damage leading to transient myoglobinuria and increased risk of kidney failure. McArdle disease is caused by recessive mutations in the muscle glycogen phosphorylase (PYGM) gene leading to absence of PYGM enzyme in skeletal muscle and preventing access to energy from muscle glycogen stores. There is currently no cure for McArdle disease. Using a preclinical animal model, we aimed to identify a clinically translatable and relevant therapy for McArdle disease. We evaluated the safety and efficacy of recombinant adeno-associated virus serotype 8 (rAAV8) to treat a murine model of McArdle disease via delivery of a functional copy of the disease-causing gene, Pygm. Intraperitoneal injection of rAAV8-Pygm at post-natal day 1–3 resulted in Pygm expression at 8 weeks of age, accompanied by improved skeletal muscle architecture, reduced accumulation of glycogen and restoration of voluntary running wheel activity to wild-type levels. We did not observe any adverse reaction to the treatment at 8 weeks post-injection. Thus, we have investigated a highly promising gene therapy for McArdle disease with a clear path to the ovine large animal model endemic to Western Australia and subsequently to patients.

The hunger games: sensing host arginine is essential for Leishmania parasite virulence

Arginine homeostasis in lysosomes is critical for growth and metabolism of mammalian cells. They employ a specific sensor (SLC38A9) that monitors intra-lysosome arginine sufficiency and subsequently up-regulates cellular mTORC1 activity. Lysosomes of macrophages (phagolysosomes) are the niche where the parasitic protozoan Leishmania resides and causes important human disease. Several years ago, we discovered that upon arginine starvation, cultured Leishmania parasites promptly activate a MAPK2-mediated Arginine Deprivation Response (ADR) pathway, resulting in up-regulation of the Leishmania arginine transporter (AAP3), as well as a small group of other transporters. Significantly, ADR is also activated during macrophage infection, implying that the intracellular parasite actively depletes arginine within the host phagolysosome, likely to prevent mTORC1 activation and enhance intracellular development. We hypothesize that ADR-mediated up-regulation of AAP3 activity is necessary to withstand the resultant arginine starvation. Both copies of the AAP3 genes are located (in tandem) on a tetrasomic chromosome (chr31), but only one (AAP3.2) is responsive to arginine deprivation. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport (mediated by AAP3.1), but lack a functional copy of AAP3.2 and are therefore not responsive to arginine starvation. While these mutants grow normally in culture as promastigotes, they were impaired in their ability to develop inside THP1 macrophages grown under physiological concentrations of arginine (0.1 mM). However, flooding the macrophage growth medium with arginine (1.5 mM) restored parasite infectivity and intracellular growth to that of wild type. The results indicate that inside the host macrophage, Leishmania must overcome the arginine “Hunger Games” by up-regulating transport of arginine via the ADR. Furthermore, the AAP3.2 mutants were ~70-80% less virulent in Balb/C mice, showing, for the first time, that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.

Signatures of relaxed selection in the CYP8B1 gene of birds and mammals

SummaryThe CYP8B1 gene is known to catalyse reactions that determine the ratio of primary bile salts and the loss of this gene has recently been linked to lack of cholic acid in the bile of naked-mole rats, elephants and manatees using forward genomics approaches. We screened the CYP8B1 gene sequence of more than 200 species and test for relaxation of selection along each terminal branch. The need for retaining a functional copy of the CYP8B1 gene is established by the presence of a conserved open reading frame across most species screened in this study. Interestingly, the dietary switch from bovid to cetacean species is accompanied by an exceptional ten amino-acid extension at the C-terminal end through a single base frame-shift deletion. We also verify that the coding frame disrupting mutations previously reported in the elephant are correct, are shared by extinct Elephantimorpha species and coincide with the dietary switch to herbivory. Relaxation of selection in the CYP8B1 gene of the wombat (Vombatus ursinus) also corresponds to drastic change in diet. In summary, our forward genomics based screen of bird and mammal species identifies recurrent changes in the selection landscape of the CYP8B1 gene concomitant with a change in dietary lipid content.

CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency

A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in Sim1 and Mc4r heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the Sim1 promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in Sim1 heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in Sim1 and Mc4r haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.