Creation of de novo cryptic splicing for ALS and FTD precision medicine.

Loss of function of the RNA-binding protein TDP-43 (TDP-LOF) is a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Here we describe TDP-REG, which exploits the specificity of cryptic splicing induced by TDP-LOF to drive protein expression when and where the disease process occurs. The SpliceNouveau algorithm combines deep learning with rational design to generate customizable cryptic splicing events within protein-coding sequences. We demonstrate that expression of TDP-REG reporters is tightly coupled to TDP-LOF in vitro and in vivo. TDP-REG enables genomic prime editing to ablate the UNC13A cryptic donor splice site specifically upon TDP-LOF. Finally, we design TDP-REG vectors encoding a TDP-43/Raver1 fusion protein that rescues key pathological cryptic splicing events, paving the way for the development of precision therapies for TDP43-related disorders.

Creation of de novo cryptic splicing for ALS/FTD precision medicine.

A system enabling the expression of therapeutic proteins specifically in diseased cells would be transformative, providing greatly increased safety and the possibility of pre-emptive treatment. Here we describe "TDP-REG", a precision medicine approach primarily for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which exploits the cryptic splicing events that occur in cells with TDP-43 loss-of-function (TDP-LOF) in order to drive expression specifically in diseased cells. In addition to modifying existing cryptic exons for this purpose, we develop a deep-learning-powered algorithm for generating customisable cryptic splicing events, which can be embedded within virtually any coding sequence. By placing part of a coding sequence within a novel cryptic exon, we tightly couple protein expression to TDP-LOF. Protein expression is activated by TDP-LOF in vitro and in vivo, including TDP-LOF induced by cytoplasmic TDP-43 aggregation. In addition to generating a variety of fluorescent and luminescent reporters, we use this system to perform TDP-LOF-dependent genomic prime editing to ablate the UNC13A cryptic donor splice site. Furthermore, we design a panel of tightly gated, autoregulating vectors encoding a TDP-43/Raver1 fusion protein, which rescue key pathological cryptic splicing events. In summary, we combine deep-learning and rational design to create sophisticated splicing sensors, resulting in a platform that provides far safer therapeutics for neurodegeneration, potentially even enabling preemptive treatment of at-risk individuals.

Genetic therapeutic advancements for Dravet Syndrome.

Dravet Syndrome is a genetic epileptic syndrome characterized by severe and intractable seizures associated with cognitive, motor, and behavioral impairments. The disease is also linked with increased mortality mainly due to sudden unexpected death in epilepsy. Over 80% of cases are due to a de novo mutation in one allele of the SCN1A gene, which encodes the α-subunit of the voltage-gated ion channel NaV1.1. Dravet Syndrome is usually refractory to antiepileptic drugs, which only alleviate seizures to a small extent. Viral, non-viral genetic therapy, and gene editing tools are rapidly enhancing and providing new platforms for more effective, alternative medicinal treatments for Dravet syndrome. These strategies include gene supplementation, CRISPR-mediated transcriptional activation, and the use of antisense oligonucleotides. In this review, we summarize our current knowledge of novel genetic therapies that are currently under development for Dravet syndrome.

Re-structuring lentiviral vectors to express genomic RNA via cap-dependent translation.

Lentiviral (LV) vectors based on human immunodeficiency virus type I (HIV-1) package two copies of their single-stranded RNA into vector particles. Normally, this RNA genome is reverse transcribed into a double-stranded DNA provirus that integrates into the cell genome, providing permanent gene transfer and long-term expression. Integration-deficient LV vectors have been developed to reduce the frequency of genomic integration and thereby limit their persistence in dividing cells. Here, we describe optimization of a reverse-transcriptase-deficient LV vector, which enables direct translation of LV RNA genomes upon cell entry, for transient expression of vector payloads as mRNA without a DNA intermediate. We have engineered a novel LV genome arrangement in which HIV-1 sequences are removed from the 5' end, to enable ribosomal entry from the 5' 7-methylguanylate cap for efficient translation of the vector payload. We have shown that this LV-mediated mRNA delivery platform provides transient transgene expression in vitro and in vivo. This has a potential application in gene and cell therapy scenarios requiring temporary payload expression in cells and tissues that can be targeted with pseudotyped LV vectors.

Cervical Gene Delivery of the Antimicrobial Peptide, Human ß-Defensin (HBD)-3, in a Mouse Model of Ascending Infection-Related Preterm Birth.

Approximately 40% of preterm births are preceded by microbial invasion of the intrauterine space; ascent from the vagina being the most common pathway. Within the cervical canal, antimicrobial peptides and proteins (AMPs) are important components of the cervical barrier which help to prevent ascending vaginal infection. We investigated whether expression of the AMP, human ß-defensin-3 (HBD3), in the cervical mucosa of pregnant mice could prevent bacterial ascent from the vagina into the uterine cavity. An adeno-associated virus vector containing both the HBD3 gene and GFP transgene (AAV8 HBD3.GFP) or control AAV8 GFP, was administered intravaginally into E13.5 pregnant mice. Ascending infection was induced at E16.5 using bioluminescent Escherichia coli (E. coli K1 A192PP-lux2). Bioluminescence imaging showed bacterial ascent into the uterine cavity, inflammatory events that led to premature delivery and a reduction in pups born alive, compared with uninfected controls. Interestingly, a significant reduction in uterine bioluminescence in the AAV8 HBD3.GFP-treated mice was observed 24 h post-E. coli infection, compared to AAV8 GFP treated mice, signifying reduced bacterial ascent in AAV8 HBD3.GFP-treated mice. Furthermore, there was a significant increase in the number of living pups in AAV HBD3.GFP-treated mice. We propose that HBD3 may be a potential candidate for augmenting cervical innate immunity to prevent ascending infection-related preterm birth and its associated neonatal consequences.

Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors.

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.

Continual Conscious Bioluminescent Imaging in Freely Moving Mice.

In vivo bioluminescent imaging allows the detection of reporter gene expression in rodents in real time. Here we describe a novel technology whereby we can generate somatotransgenic rodents with the use of a viral vector carrying a luciferase transgene. We are able to achieve long term luciferase expression by a single injection of lentiviral or adeno-associated virus vectors to newborn mice. Further, we describe whole body bioluminescence imaging of conscious mice in a noninvasive manner, thus enforcing the 3R's (replacement, reduction, and refinement) of biomedical animal research.

Foamy Virus Vectors Transduce Visceral Organs and Hippocampal Structures following In Vivo Delivery to Neonatal Mice.

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders.

Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer.

Argininosuccinate lyase (ASL) belongs to the hepatic urea cycle detoxifying ammonia, and the citrulline-nitric oxide (NO) cycle producing NO. ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia, multiorgan disease and neurocognitive impairment despite treatment aiming to normalise ammonaemia without considering NO imbalance. Here we show that cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress independent of hyperammonaemia. Intravenous injection of AAV8 vector into adult or neonatal ASL-deficient mice demonstrates long-term correction of the hepatic urea cycle and the cerebral citrulline-NO cycle, respectively. Cerebral disease persists if ammonaemia only is normalised but is dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This correlates with behavioural improvement and reduced cortical cell death. Thus, neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Disease amelioration by simultaneous brain and liver gene transfer with one vector, to treat both metabolic pathways, provides new hope for hepatocerebral metabolic diseases.