-
Journal of Translational Medicine Jul 2023Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme,...
BACKGROUND
Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9.
METHODS
Here, intraparenchymal AAV was delivered in sheep using catheters or Hamilton syringes, placed using Brainlab cranial navigation for convection enhanced delivery, to reduce proximal vector expression and improve spread.
RESULTS
Hamilton syringes gave improved AAV-GFP distribution, despite lower vector doses and titres. AAV-TT-GFP displayed moderately better transduction compared to AAV9-GFP but both serotypes almost exclusively transduced neurons. Functional HGSNAT enzyme was detected in 24-37% of a 140g gyrencephalic sheep brain using AAV9-HGSNAT with three injections in one hemisphere.
CONCLUSIONS
Despite variabilities in volume and titre, catheter design may be critical for efficient brain delivery. These data help inform a clinical trial for MPSIIIC.
Topics: Animals; Acetyltransferases; Brain; Dependovirus; Disease Models, Animal; Genetic Vectors; Heparitin Sulfate; Mucopolysaccharidoses; Mucopolysaccharidosis III; Sheep; Genetic Therapy
PubMed: 37407981
DOI: 10.1186/s12967-023-04208-1 -
Journal of Thrombosis and Haemostasis :... May 2024Adeno-associated virus gene therapy has been the subject of intensive investigation for monogenic disease gene addition therapy for more than 25 years, yet few therapies... (Review)
Review
Adeno-associated virus gene therapy has been the subject of intensive investigation for monogenic disease gene addition therapy for more than 25 years, yet few therapies have been approved by regulatory agencies. Most have not progressed beyond phase 1/2 due to toxicity, lack of efficacy, or both. The liver is a natural target for adeno-associated virus since most serotypes have a high degree of tropism for hepatocytes due to cell surface receptors for the virus and the unique liver sinusoidal geometry facilitating high volumes of blood contact with hepatocyte cell surfaces. Recessive monogenic diseases such as hemophilia represent promising targets since the defective proteins are often synthesized in the liver and secreted into the circulation, making them easy to measure, and many do not require precise regulation. Yet, despite initiation of many disease-specific clinical trials, therapeutic windows are often nonexistent, resulting in excess toxicity and insufficient efficacy. Iterative progress built on these attempts is best illustrated by hemophilia, with the first regulatory approvals for factor IX and factor VIII gene therapies eventually achieved 25 years after the first gene therapy studies in humans. Although successful gene transfer may result in the production of sufficient transgenic protein to modify the disease, many emerging questions on durability, predictability, reliability, and variability of response have not been answered. The underlying biology accounting for these heterogeneous responses and the interplay between host and virus is the subject of intense investigation and the subject of this review.
Topics: Humans; Dependovirus; Hemophilia A; Genetic Therapy; Liver; Genetic Vectors; Animals; Factor VIII; Gene Transfer Techniques
PubMed: 38103734
DOI: 10.1016/j.jtha.2023.12.005 -
Nature Communications Mar 2024Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction...
Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg/Rag2/Fah/Aavr (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.
Topics: Humans; Animals; Mice; Liver; Dependovirus; Disease Models, Animal; Genetic Therapy; Hepatocytes
PubMed: 38438373
DOI: 10.1038/s41467-024-46017-0 -
Circulation Oct 2023Cardiac reprogramming is a technique to directly convert nonmyocytes into myocardial cells using genes or small molecules. This intervention provides functional benefit...
BACKGROUND
Cardiac reprogramming is a technique to directly convert nonmyocytes into myocardial cells using genes or small molecules. This intervention provides functional benefit to the rodent heart when delivered at the time of myocardial infarction or activated transgenically up to 4 weeks after myocardial infarction. Yet, several hurdles have prevented the advancement of cardiac reprogramming for clinical use.
METHODS
Through a combination of screening and rational design, we identified a cardiac reprogramming cocktail that can be encoded in a single adeno-associated virus. We also created a novel adeno-associated virus capsid that can transduce cardiac fibroblasts more efficiently than available parental serotypes by mutating posttranslationally modified capsid residues. Because a constitutive promoter was needed to drive high expression of these cell fate-altering reprogramming factors, we included binding sites to a cardiomyocyte-restricted microRNA within the 3' untranslated region of the expression cassette that limits expression to nonmyocytes. After optimizing this expression cassette to reprogram human cardiac fibroblasts into induced cardiomyocyte-like cells in vitro, we also tested the ability of this capsid/cassette combination to confer functional benefit in acute mouse myocardial infarction and chronic rat myocardial infarction models.
RESULTS
We demonstrated sustained, dose-dependent improvement in cardiac function when treating a rat model 2 weeks after myocardial infarction, showing that cardiac reprogramming, when delivered in a single, clinically relevant adeno-associated virus vector, can support functional improvement in the postremodeled heart. This benefit was not observed with GFP (green fluorescent protein) or a hepatocyte reprogramming cocktail and was achieved even in the presence of immunosuppression, supporting myocyte formation as the underlying mechanism.
CONCLUSIONS
Collectively, these results advance the application of cardiac reprogramming gene therapy as a viable therapeutic approach to treat chronic heart failure resulting from ischemic injury.
Topics: Rats; Mice; Humans; Animals; Dependovirus; Myocytes, Cardiac; Myocardial Infarction; MicroRNAs; Genetic Therapy; Green Fluorescent Proteins; Cellular Reprogramming; Fibroblasts
PubMed: 37602409
DOI: 10.1161/CIRCULATIONAHA.122.061542 -
Viruses Sep 2023The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with...
The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with optimized yield and transduction efficiency. The Baculovirus/Sf9 system is a promising platform for high yield production; however, a major drawback to using an invertebrate cell line compared to a mammalian system is a generally altered AAV capsid stoichiometry resulting in lower biological potency. Here, we introduce a term of the structural and biological "fitness" of an AAV capsid as a function of two interdependent parameters: (1) packaging efficiency (yield), and (2) transduction efficiency (infectivity). Both parameters are critically dependent on AAV capsid structural proteins VP1/2/3 stoichiometry. To identify an optimal AAV capsid composition, we developed a novel Directed Evolution (DE) protocol for assessing the structural and biological fitness of Sf9-manufactured rAAV for any given serotype. The approach involves the packaging of a combinatorial capsid library in insect Sf9 cells, followed by a library screening for high infectivity in human Cre-recombinase-expressing C12 cells. One single DE selection round, complemented by Next-Generation Sequencing (NGS) and guided by in silico analysis, identifies a small subset of VP1 translation initiation sites (known as Kozak sequence) encoding "fit" AAV capsids characterized by a high production yield and superior transduction efficiencies.
Topics: Animals; Humans; Genetic Vectors; Dependovirus; Capsid Proteins; Cell Line; Capsid; Mammals
PubMed: 37896760
DOI: 10.3390/v15101983 -
PloS One 2023Recombinant adeno-associated virus (rAAV) vectors have become a reliable strategy for delivering gene therapies. As rAAV capsid content is known to be heterogeneous,...
Recombinant adeno-associated virus (rAAV) vectors have become a reliable strategy for delivering gene therapies. As rAAV capsid content is known to be heterogeneous, methods for rAAV characterization are critical for assessing the efficacy and safety of drug products. Multiplex digital PCR (dPCR) has emerged as a popular molecular approach for characterizing capsid content due to its high level of throughput, accuracy, and replicability. Despite growing popularity, tools to accurately analyze multiplexed data are scarce. Here, we introduce a novel statistical model to estimate genome integrity from duplex dPCR assays. This work demonstrates that use of a Poisson-multinomial mixture distribution significantly improves the accuracy and quantifiable range of duplex dPCR assays over currently available models.
Topics: Genetic Vectors; Genetic Therapy; Transgenes; Polymerase Chain Reaction; Dependovirus
PubMed: 38096204
DOI: 10.1371/journal.pone.0293277 -
Journal of Veterinary Internal Medicine 2023A treatment of chronic kidney disease (CKD)-associated anemia in cats is needed. SB-001 is an adeno-associated virus-vectored (AAV)-based gene therapeutic agent that is...
BACKGROUND
A treatment of chronic kidney disease (CKD)-associated anemia in cats is needed. SB-001 is an adeno-associated virus-vectored (AAV)-based gene therapeutic agent that is administered intramuscularly, causing the expression of feline erythropoietin.
HYPOTHESIS/OBJECTIVE
We hypothesized that SB-001 injection would lead to a sustained increase in PCV in cats with CKD-associated anemia.
ANIMALS
Twenty-three cats with International Renal Interest Society (IRIS) Stage 2 to 4 CKD-associated anemia were enrolled at 4 veterinary clinics.
METHODS
In a prospective clinical trial, cats were treated with 1 of 3 regimens of SB-001 (Lo 1.2 × 10 genome copies [GCs] on Day 0; Lo ± Hi [supplemental 2nd dose of 3.65 × 10 GC on Day 42]; Hi 3.65 × 10 GC IM on Day 0) and followed for 70 days.
RESULTS
A response to SB-001 at any time between Day 28 and Day 70 was seen in 86% (95% confidence interval 65, 97%) of all cats. There was a significant (P < .003) increase in PCV from Day 0 to Day 28 (mean increase 6 ± 6 percentage points [pp]; n = 21), Day 42 (8 ± 9 pp; n = 21), Day 56 (10 ± 11 pp; n = 17), and Day 70 (13 ± 14 pp, n = 14). Twelve cats were hypertensive at baseline, 4 of which developed encephalopathy during the study. An additional 6 cats became hypertensive during the study.
CONCLUSIONS AND CLINICAL IMPORTANCE
Results of this study suggest that SB-001 therapy represents a suitable single injection treatment that can address nonregenerative anemia in cats with CKD. It was generally well tolerated; however, hypertension and encephalopathy developed in some cats as previously described in association with erythropoiesis-stimulating agent therapy.
Topics: Cats; Animals; Dependovirus; Prospective Studies; Renal Insufficiency, Chronic; Anemia; Erythropoietin; Hypertension; Brain Diseases; Genetic Therapy; Cat Diseases
PubMed: 37847024
DOI: 10.1111/jvim.16900 -
CNS Neuroscience & Therapeutics Mar 2024Epilepsy is a widespread and chronic disease of the central nervous system caused by a variety of factors. Mitochondrial ferritin (FtMt) refers to ferritin located...
Mitochondrial ferritin alleviates ferroptosis in a kainic acid-induced mouse epilepsy model by regulating iron homeostasis: Involvement of nuclear factor erythroid 2-related factor 2.
BACKGROUND
Epilepsy is a widespread and chronic disease of the central nervous system caused by a variety of factors. Mitochondrial ferritin (FtMt) refers to ferritin located within the mitochondria that may protect neurons against oxidative stress by binding excess free iron ions in the cytoplasm. However, the potential role of FtMt in epilepsy remains unclear. We aimed to investigate whether FtMt and its related mechanisms can regulate epilepsy by modulating ferroptosis.
METHODS
Three weeks after injection of adeno-associated virus (AAV) in the skull of adult male C57BL/6 mice, kainic acid (KA) was injected into the hippocampus to induce seizures. Primary hippocampal neurons were transfected with siRNA using a glutamate-mediated epilepsy model. After specific treatments, Western blot analysis, immunofluorescence, EEG recording, transmission electron microscopy, iron staining, silver staining, and Nissl staining were performed.
RESULTS
At different time points after KA injection, the expression of FtMt protein in the hippocampus of mice showed varying degrees of increase. Knockdown of the FtMt gene by AAV resulted in an increase in intracellular free iron levels and a decrease in the function of iron transport-related proteins, promoting neuronal ferroptosis and exacerbating epileptic brain activity in the hippocampus of seizure mice. Additionally, increasing the expression level of FtMt protein was achieved by AAV-mediated upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) gene in the hippocampus of seizure mice.
CONCLUSIONS
In epilepsy, Nrf2 modulates ferroptosis by involving the expression of FtMt and may be a potential therapeutic mechanism of neuronal injury after epilepsy. Targeting this relevant process for treatment may be a therapeutic strategy to prevent epilepsy.
Topics: Male; Animals; Mice; Mice, Inbred C57BL; Kainic Acid; Ferroptosis; NF-E2-Related Factor 2; Epilepsy; Seizures; Glutamic Acid; Dependovirus; Disease Models, Animal; Ferritins; Homeostasis
PubMed: 38439636
DOI: 10.1111/cns.14663 -
Nature Communications May 2024Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a...
Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.
Topics: Animals; Gene Editing; Female; Dependovirus; Mice; Herpesvirus 1, Human; Herpes Simplex; Viral Load; Virus Shedding; Disease Models, Animal; Virus Latency; Humans; Genetic Vectors; Vero Cells; Genetic Therapy; Herpes Genitalis; DNA, Viral
PubMed: 38740820
DOI: 10.1038/s41467-024-47940-y -
Microbial Biotechnology Oct 2023During the COVID-19 pandemic, two further novel viral epidemics were described in 2022, monkeypox virus infections in men having sex with men and non-A to E hepatitis in...
During the COVID-19 pandemic, two further novel viral epidemics were described in 2022, monkeypox virus infections in men having sex with men and non-A to E hepatitis in children. The latter occurred in the first half of 2022 with about 1000 cases worldwide, necessitating liver transplantation in 5% and causing death in 2% of patients. It took some effort to clarify the cause of the novel hepatitis epidemic. Researchers were confronted with a polymicrobial viral infection consisting of an adenovirus-associated virus type 2 (AAV2) infection, co-occurring with either human adenovirus type 41 (HAdV41) or herpesvirus infections; most prominently human herpesvirus type 6 (HHV-6). AAV-2, a small Dependovirus of the Parvovirus family, needs these helper viruses for its replication. AAV2 is used as a vector for liver-targeting gene therapy but was not previously known to cause acute hepatitis. HAdV41 and HHV-6 are mostly known to cause diarrhoea and febrile illnesses associated with skin rashes in children, respectively. Except for a few case reports of HHV-6 hepatitis, HAdV and HHV-6 are mostly known as major pathogens in immunosuppressed transplantation patients. A potential role of SARS-CoV-2 has also been discussed but the most popular hypothesis involves an indirect role of the COVID-19 pandemic for this novel disease. Exposure to HHV-6 infections occurs nearly quantitatively during the first year of life. Social distancing measures, followed by the lifting of these measures in 2022 might have caused a delayed exposure to multiple, normally benign childhood viral infections eliciting a dysregulated immune response with pathological effects for liver cells. In the fall of 2022, when these conditions were not longer met, case numbers dwindled. The hypothesis of an unequilibrated immune response instead of intrinsic cytopathic activity of the implicated viruses is further supported by the enrichment of a particular HLA allele in cases over controls.
Topics: Male; Humans; Child; Pandemics; COVID-19; SARS-CoV-2; Virus Diseases; Roseolovirus Infections
PubMed: 37602673
DOI: 10.1111/1751-7915.14329