Secreted therapeutics: monitoring durability of microRNA-based gene therapies in the central nervous system.

The preclinical development of microRNA-based gene therapies for inherited neurodegenerative diseases is accompanied by translational challenges. Due to the inaccessibility of the brain to periodically evaluate therapy effects, accessible and reliable biomarkers indicative of dosing, durability and therapeutic efficacy in the central nervous system are very much needed. This is particularly important for viral vector-based gene therapies, in which a one-time administration results in long-term expression of active therapeutic molecules in the brain. Recently, extracellular vesicles have been identified as carriers of RNA species, including microRNAs, and proteins in all biological fluids, whilst becoming potential sources of biomarkers for diagnosis. In this study, we investigated the secretion and potential use of circulating miRNAs associated with extracellular vesicles as suitable sources to monitor the expression and durability of gene therapies in the brain. Neuronal cells derived from induced pluripotent stem cells were treated with adeno-associated viral vector serotype 5 carrying an engineered microRNA targeting huntingtin or ataxin3 gene sequences, the diseases-causing genes of Huntington disease and spinocerebellar ataxia type 3, respectively. After treatment, the secretion of mature engineered microRNA molecules was confirmed, with extracellular microRNA levels correlating with viral dose and cellular microRNA expression in neurons. We further investigated the detection of engineered microRNAs over time in the CSF of non-human primates after a single intrastriatal injection of adeno-associated viral vector serotype 5 carrying a huntingtin-targeting engineered microRNA. Quantifiable engineered microRNA levels enriched in extracellular vesicles were detected in the CSF up to 2 years after brain infusion. Altogether, these results confirm the long-term expression of adeno-associated viral vector serotype 5-delivered microRNAs and support the use of extracellular vesicle-associated microRNAs as novel translational pharmacokinetic markers in ongoing clinical trials of gene therapies for neurodegenerative diseases.

Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients.

The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases. RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ∼50% reduction of nuclear RNA foci in (G4C2)44-expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients.

Adeno-associated virus liver transduction efficiency measured by in vivo [18F]FHBG positron emission tomography imaging in rodents and nonhuman primates.

Recombinant adeno-associated virus 5 (rAAV5) represents a candidate vector with unique advantages for the treatment of hepatic disorders because of its narrow hepatic tropism. Noninvasive in vivo imaging of transgene expression provides an important tool with which to quantify the transduction efficiency, and duration and location, of transgene expression. In this study, we used positron emission tomography (PET) and positron emission tomography-computed tomography (PET-CT) imaging to monitor liver transduction efficacy in rodents and nonhuman primates that received rAAV5 vector encoding herpes simplex virus thymidine kinase (HSV-TK). HSV-TK expression in liver was also measured by immunohistochemistry. Notable differences in liver transduction efficiency were found, dependent on the animal species and sex. Male rodents were better transduced than females, as previously described. Moreover, male nonhuman primates also displayed increased hepatic expression of the rAAV5-delivered transgene, indicating that differences in rAAV-mediated liver transduction can be anticipated in humans. Our results demonstrate the high sensitivity and reproducibility of PET, using HSV-TK and [(18)F]FHBG, to detect gene expression after rAAV vector administration into living animals, confirming the utility of this technology in the quantification of transgene expression, even at low expression levels. However, we also describe how an immune response against HSV-TK hampered analysis of long-term expression in nonhuman primates.

Sustained enzymatic correction by rAAV-mediated liver gene therapy protects against induced motor neuropathy in acute porphyria mice.

Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.

AAV gene therapy as a means to increase apolipoprotein (Apo) A-I and high-density lipoprotein-cholesterol levels: correction of murine ApoA-I deficiency.

BACKGROUND: Inherited apolipoprotein (Apo) A-I deficiency is an orphan disorder characterized by high-density lipoprotein (HDL)-cholesterol deficiency and premature atherosclerosis. Constitutive over-expression of ApoA-I might provide a means to treat this disease. The present study provides a comprehensive evaluation of adeno-associated virus (AAV)-mediated ApoA-I gene delivery to express human (h)ApoA-I and correct the low HDL-cholesterol phenotype associated with ApoA-I deficiency. METHODS: In an effort to maximize AAV-mediated gene expression, we performed head-to-head comparisons of recombinant AAVs with pseudotype capsids 1, 2, 6 and 8 administered by different routes with the use of five different liver-specific promoters in addition to cytomegalovirus as single-stranded or as self-complementary (sc) AAV vectors. RESULTS: Intravenous administration of 1 x 10(13) gc/kg scAAV8, in combination with the liver-specific promoter LP1, in female ApoA-I(-/-) mice resulted in hApoA-I expression levels of 634 +/- 69 mg/l, which persisted for the duration of the study (15 weeks). This treatment resulted in full recovery of HDL-cholesterol levels with correction of HDL particle size and apolipoprotein composition. In addition, we observed increased adrenal cholesterol content and a significant increase in bodyweight in treated mice. CONCLUSIONS: The present study demonstrates that systemic delivery of a scAAV8 vector provides a means for efficient liver expression of hApoA-I, thereby correcting the lipid abnormalities associated with murine ApoA-I deficiency. Importantly, the study demonstrates that AAV-based gene therapy can be used to express therapeutic proteins at a high level for a prolonged period of time and, as such, provides a basis for further development of this strategy to treat hApoA-I deficiency.

Neonatal basolateral amygdala lesions affect monoamine and cannabinoid brain systems in adult rats.

There is evidence for neurodevelopment disturbances in schizophrenia. In rats, a neonatal basolateral amygdala lesion induces behavioural features in adults reminiscent of the symptomatology of schizophrenia. Dopamine plays a key role in the pathogenesis of schizophrenia, and cannabis use has been implicated in the risk for developing schizophrenia. The effects of an excitotoxic, bilateral basolateral amygdala lesion on postnatal days 7 or 21 were compared when the rats were adult. The behavioural response to a novelty challenge and the level of dopamine receptors and cannabinoid receptors in the brain using in-vitro autoradiography was determined. In brain tissue punches concentrations of monoamines and metabolites were determined by high-performance liquid chromatography. The neonatal lesion, but not the later lesion induced behavioural hyperactivity and biochemical effects. The neonatal lesion reduced the density of dopamine D2-like, but not D3-, and less markely D1-like receptors and increased dopamine turnover. These effects were observed in the mesolimbic, but not in the striatal regions. In contrast, density of cannabinoid receptors was increased in the striatal, but not the mesolimbic regions of these animals. Noradrenergic neurotransmission was reduced in both regions. The present findings contribute to the idea that the neonatal basolateral amygdala lesion induces features in adults reminiscent of the neurodevelopmental disturbances in schizophrenia, with a focus on the amygdala-prefrontal cortex-nucleus accumbens circuit.

Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.

Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is low. We routinely induce cardiomyocyte differentiation of the HES-2 cell line by coculture with a visceral endoderm-like cell line, END-2, in the presence of 20% fetal calf serum (FCS). In this study, we demonstrate a striking inverse relationship between cardiomyocyte differentiation and the concentration of FCS during HES-2-END-2 coculture. The number of beating areas in the cocultures was increased 24-fold in the absence of FCS compared with the presence of 20% FCS. An additional 40% increase in the number of beating areas was observed when ascorbic acid was added to serum-free cocultures. The increase in serum-free cocultures was accompanied by increased mRNA and protein expression of cardiac markers and of Isl1, a marker of cardiac progenitor cells. The number of beating areas increased up to 12 days after initiation of coculture of HES-2 with END-2 cells. However, the number of alpha-actinin-positive cardiomyocytes per beating area did not differ significantly between serum-free cocultures (503 +/- 179; mean +/- standard error of the mean) and 20% FCS cocultures (312 +/- 227). The stimulating effect of serum-free coculture on cardiomyocyte differentiation was observed not only in HES-2 but also in the HES-3 and HES-4 cell lines. To produce sufficient cardiomyocytes for cell replacement therapy in the future, upscaling cardiomyocyte formation from hESCs is essential. The present data provide a step in this direction and represent an improved in vitro model, without interfering factors in serum, for testing other factors that might promote cardiomyocyte differentiation.

Effects of neonatal amygdala lesions on [125I] neurotensin binding in specific brain areas of adult rat.

Neurotensin has been implicated in the pathophysiology of schizophrenia. The neonatal amygdala lesion in rat has been proposed to be a neurodevelopmental model for some aspects of schizophrenia. [125I] Neurotensin binding was assessed in adult rats using in vitro autoradiography following a lesion of the basolateral amygdala at postnatal day 7 (Pd 7) or postnatal day 21 (Pd 21). The Pd 7 and Pd 21 lesions differentially affected neurotensin receptor densities in the hippocampal complex and (less pronounced) in the dopaminergic cell regions, implying a neurodevelopmental cause. These results may be of relevance for the involvement of neurotensin in the pathogenesis of schizophrenia.