Neospora caninum is an obligate intracellular protozoan that affects several animal species. It is not pathogenic for humans, and its ability to infect and lyse a variety of cells and stimulate the immune system makes it...Neospora caninum is an obligate intracellular protozoan that affects several animal species. It is not pathogenic for humans, and its ability to infect and lyse a variety of cells and stimulate the immune system makes it an interesting drug candidate in oncology. The intrinsic oncolytic properties of N. caninum have been confirmed in several preclinical models. Moreover, it can be modified to improve its safety and/or efficacy against cancer cells. In this study, we propose the legal categorization of this new biological drug candidate and the impact of modifications, notably the integration of a suicide gene, the deletion of a gene allowing its multiplication in healthy cells, and/or the insertion of a gene coding for a therapeutic protein into its genome. When unmodified, N. caninum can be categorized as a biological medicinal product, whereas modifications aimed at increasing its safety classify it as a Somatic Cell Therapy Medicinal Product, and modifications aiming to increase its efficacy or both safety and efficacy make it as a Gene Therapy Medicinal Product. This categorization is fundamental because it determines the guidelines applicable for preclinical development. These guidelines being numerous and complex, we have focused on the key requirements necessary for the development of the future medicinal product.
McColl-Carboni A, Dollive S, Laughlin S
… +16 more, Lushi R, MacArthur M, Zhou S, Gagnon J, Smith CA, Burnham B, Horton R, Lata D, Uga B, Natu K, Michel E, Slater C, DaSilva E, Bruccoleri R, Kelly T, McGivney JB
Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on s...Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on safety and efficacy of AAV products, they are generally considered impurities because they are not the intended fully intact vector product. The presence of these impurities could impact product efficacy due to potential competition with fully packaged AAVs for cellular transduction, as well as have potential implications to patient safety due to increased capsid load during dosing. To determine the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro (infectivity, gene expression, biological activity) and in vivo potency assays to determine potency as a function of capsid content, our results indicate that while intermediate capsids contribute to the vector genome titer of the product and are equally as infectious as full capsids, they do not contribute to the potency of the AAV product. This study confirms the criticality of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product.
Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure impacts gene therapy eligibility. The aim of this multi...Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure impacts gene therapy eligibility. The aim of this multicenter epidemiologic study was to determine the prevalence and persistence of preexisting immunity against AAV2, AAV5, and AAV8, in adult participants with hemophilia A or B. Blood samples were collected at baseline and annually for ≤3 years at trial sites in Austria, France, Germany, Italy, Spain, and the United States. At baseline, AAV8, AAV2, and AAV5 neutralizing antibodies (NAbs) were present in 46.9%, 53.1%, and 53.4% of participants, respectively; these values remained stable at Years 1 and 2. Co-prevalence of NAbs to at least two serotypes and all three serotypes was present at baseline for ~40% and 38.2% of participants, respectively. For each serotype, ~10% of participants who tested negative for NAbs at baseline were seropositive at Year 1. At baseline, 38.3% of participants had detectable cell mediated immunity by ELISpot, although no correlations were observed with the humoral response. In conclusion, participants with hemophilia may have significant preexisting immunity to AAV capsids. Insights from this study may assist in understanding capsid-based immunity trends in participants considering AAV vector-based gene therapy.
Patients with sialidosis (mucolipidosis type I) type I typically present with myoclonus, seizures, ataxia, cherry-red spots, and blindness because of mutations in the neuraminidase 1 (NEU1) gene. Currently, there is no t...Patients with sialidosis (mucolipidosis type I) type I typically present with myoclonus, seizures, ataxia, cherry-red spots, and blindness because of mutations in the neuraminidase 1 (NEU1) gene. Currently, there is no treatment for sialidosis. In this study, we developed an adeno-associated virus (AAV)-mediated gene therapy for a Neu1 knockout (Neu1) mouse model of sialidosis. The vector, AAV9-P3-NP, included the human NEU1 promoter, NEU1 cDNA, IRES, and CTSA cDNA. Untreated Neu1 mice showed astrogliosis and microglial LAMP1 accumulation in the nervous system, including brain, spinal cord, and dorsal root ganglion, together with impaired motor function. Coexpression of NEU1 and protective protein/cathepsin A (PPCA) in neonatal Neu1 mice by intracerebroventricular injection, and less effective by facial vein injection, decreased astrogliosis and LAMP1 accumulation in the nervous system and improved rotarod performance of the treated mice. Facial vein injection also improved the grip strength and survival of Neu1 mice. Therefore, cerebrospinal fluid delivery of AAV9-P3-NP, which corrects the neurological deficits of mice with sialidosis, could be a suitable treatment for patients with sialidosis type I. After intracerebroventricular or facial vein injection of AAV vectors, NEU1 and PPCA are expressed together. PPCA-protected NEU1 is then sent to lysosomes, where β-Gal binds to this complex to form a multienzyme complex in order to execute its function.
Baculoviruses have shown great potential as gene delivery vectors in mammals, although their effectiveness in transferring genes varies across different cell lines. A widely employed strategy to improve transduction effi...Baculoviruses have shown great potential as gene delivery vectors in mammals, although their effectiveness in transferring genes varies across different cell lines. A widely employed strategy to improve transduction efficiency is the pseudotyping of viral vectors. In this study, we aimed to develop a stable Sf9 insect cell line that inducibly expresses the G-protein of the vesicular stomatitis virus to pseudotype budded baculoviruses. It was obtained by inserting the VSV-G gene under the control of the very strong and infection-inducible p promoter and was subsequently diluted to establish oligoclonal lines, which were selected by the fusogenic properties of VSV-G and its expression levels in infected cells and purified budded virions. Next, to enhance the performance of the cell line, the infection conditions under which functional pseudotyped baculoviruses are obtained were optimized. Finally, different baculoviruses were pseudotyped and the expression of the transgene was quantified in mammalian cells of diverse origins using flow cytometry. The transduction efficiency of pseudotyped baculovirus consistently increased across all tested mammalian cell lines compared with control viruses. These findings demonstrate the feasibility and advantages of improving gene delivery performance without the need to insert the pseudotyping gene into the baculoviral genomes.
Retinitis pigmentosa (RP) is a heterogeneous disease and the main cause of vision loss within the group of inherited retinal diseases (IRDs). IRDs are a group of rare disorders caused by mutations in one or more of over...Retinitis pigmentosa (RP) is a heterogeneous disease and the main cause of vision loss within the group of inherited retinal diseases (IRDs). IRDs are a group of rare disorders caused by mutations in one or more of over 280 genes which ultimately result in blindness. Modifier genes play a key role in modulating disease phenotypes, and mutations in them can affect disease outcomes, rate of progression, and severity. Our previous studies have demonstrated that the nuclear hormone receptor 2 family e, member 3 (Nr2e3) gene reduced disease progression and loss of photoreceptor cell layers in Rho mice. This follow up, pharmacology study evaluates a longitudinal NR2E3 dose response in the clinically relevant heterozygous Rho mouse. Reduced retinal degeneration and improved retinal morphology was observed 6 months following treatment evaluating three different NR2E3 doses. Histological and immunohistochemical analysis revealed regions of photoreceptor rescue in the treated retinas of Rho mice. Functional assessment by electroretinogram (ERG) showed attenuated photoreceptor degeneration with all doses. This study demonstrates the effectiveness of different doses of NR2E3 at reducing retinal degeneration and informs dose selection for clinical trials of Rho-associated RP.
Burdo TH, Chen C, Kaminski R
… +18 more, Sariyer IK, Mancuso P, Donadoni M, Smith MD, Sariyer R, Caocci M, Liao S, Liu H, Huo W, Zhao H, Misamore J, Lewis MG, Simonyan V, Thompson EE, Xu EY, Cradick TJ, Gordon J, Khalili K
Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show t...Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken β-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5' and 3' ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.
Gene therapy products (GTPs) used for incurable diseases can be expedited for early commercialization to fulfill unmet needs. This study analyzed the expedited programs available for GTPs in the US, EU, Japan, and South...Gene therapy products (GTPs) used for incurable diseases can be expedited for early commercialization to fulfill unmet needs. This study analyzed the expedited programs available for GTPs in the US, EU, Japan, and South Korea using their regulatory authorities' websites, related regulations, and documents. In total, there were five expedited programs available for GTPs in the US, four in the EU, and three in both Japan and South Korea, of which four are tailored for GTPs. These programs, sharing similar objectives, can be categorized as those expediting drug development, review, and approval. However, variations are observed in eligibility criteria, specific benefits, and post-marketing study conditions across regulatory authorities. Additionally, the criteria for orphan drug designation for a rare disease differs in prevalence thresholds, incentive offered, and marketing exclusivity period. Overall, 19 GTPs were approved-13 in the US, 14 in the EU, eight in Japan, and three in South Korea-with majority obtaining regulatory approval through at least one expedited program. Therefore, future studies can analyze whether acquiring multiple expedited programs accelerates the drug development and commercialization of GTPs compared with when only one expedited program is processed. Additionally, inter-authority scientific discussion is encouraged for harmonization of expedited program requirements.
Ghrelin is commonly known as the 'hunger hormone' due to its role in stimulating food intake in humans. However, the roles of ghrelin extend beyond regulating hunger. Our aim was to investigate the ability of ghrelin to...Ghrelin is commonly known as the 'hunger hormone' due to its role in stimulating food intake in humans. However, the roles of ghrelin extend beyond regulating hunger. Our aim was to investigate the ability of ghrelin to protect against hydrogen peroxide (HO), a reactive oxygen species commonly associated with cardiac injury. An in vitro model of oxidative stress was developed using HO injured H9c2 cells. Despite lentiviral ghrelin overexpression, H9c2 cell viability and mitochondrial function were not protected following HO injury. We found that H9c2 cells lack expression of the preproghrelin cleavage enzyme prohormone convertase 1 (encoded by PCSK1), required to convert ghrelin to its active form. In contrast, we found that primary rat cardiomyocytes do express PCSK1 and were protected from HO injury by lentiviral ghrelin overexpression. In conclusion, we have shown that ghrelin expression can protect primary rat cardiomyocytes against HO, though this effect was not observed in other cell types tested.
Base editors are a type of double-stranded break (DSB)-free gene editing technology that has opened up new possibilities for precise manipulation of mitochondrial DNA (mtDNA). This includes cytosine and adenosine base ed...Base editors are a type of double-stranded break (DSB)-free gene editing technology that has opened up new possibilities for precise manipulation of mitochondrial DNA (mtDNA). This includes cytosine and adenosine base editors and more recently guanosine base editors. Because of having low off-target and indel rates, there is a growing interest in developing and evolving this research field. Here, we provide a detailed update on DNA base editors. While base editing has widely been used for nuclear genome engineering, the growing interest in applying this technology to mitochondrial DNA has been faced with several challenges. While Cas9 protein has been shown to enter mitochondria, use of smaller Cas proteins, such as Cas12a, has higher import efficiency. However, sgRNA transfer into mitochondria is the most challenging step. sgRNA structure and ratio of Cas protein to sgRNA are both important factors for efficient sgRNA entry into mitochondria. In conclusion, while there are still several challenges to be addressed, ongoing research in this field holds the potential for new treatments and therapies for mitochondrial disorders.
EPM1 is the most common form of Progressive Myoclonus Epilepsy characterized by late-childhood onset, ever-worsening and disabling myoclonus, seizures, ataxia, psychiatric disease, and shortened lifespan. EPM1 is caused...EPM1 is the most common form of Progressive Myoclonus Epilepsy characterized by late-childhood onset, ever-worsening and disabling myoclonus, seizures, ataxia, psychiatric disease, and shortened lifespan. EPM1 is caused by expansions of a dodecamer repeat sequence in the promoter of CSTB (cystatin B), which dramatically reduces, but does not eliminate, gene expression. The relatively late onset and consistent presence of a minimal amount of protein product makes EPM1 a favorable target for gene replacement therapy. If treated early, these children's normally developed brains could be rescued from the neurodegeneration that otherwise follows, and their cross-reactive immunological material (CRIM) positive status greatly reduces transgene related toxicity. We performed a proof-of-concept CSTB gene replacement study in Cstb knockout mice by introducing full-length human CSTB driven by the CBh promoter packaged in AAV9 and administered at postnatal days 21 and 60. Mice were sacrificed at 2 or 9 months of age, respectively. We observed significant improvements in expression levels of neuroinflammatory pathway genes and cerebellar granule cell layer apoptosis, as well as amelioration of motor impairment. The data suggest that gene replacement is a promising therapeutic modality for EPM1 and could spare affected children and families the ravages of this otherwise severe neurodegenerative disease.
The adeno-associated virus (AAV) gene therapy has been widely applied to mouse models for deafness. But, AAVs could transduce non-targeted organs after inner ear delivery due to their low cell-type specificity. This stud...The adeno-associated virus (AAV) gene therapy has been widely applied to mouse models for deafness. But, AAVs could transduce non-targeted organs after inner ear delivery due to their low cell-type specificity. This study compares transgene expression and biodistribution of AAV1, AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB after round window membrane (RWM) injection in neonatal mice. The highest virus concentration was detected in the injected cochlea. AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB transduced both inner hair cells (IHCs) and outer hair cells (OHCs) with high efficiency, while AAV1 transduced IHCs with high efficiency but OHCs with low efficiency. All AAV subtypes finitely transduced contralateral inner ear, brain, heart, and liver compared with the injected cochlea. In most brain regions, the enhanced green fluorescent protein (eGFP) expression of AAV1 and AAV2 was lower than that of other four subtypes. We suggested the cochlear aqueduct might be one of routes for vectors instantaneously infiltrating into the brain from the cochlea through a dye tracking test. In summary, our results provide available data for further investigating the biodistribution of vectors through local inner ear injection and afford a reference for selecting AAV serotypes for gene therapy toward deafness.
Small promoters capable of driving potent neuron-restricted gene expression are required to support successful brain circuitry and clinical gene therapy studies. However, converting large promoters into functional MiniPr...Small promoters capable of driving potent neuron-restricted gene expression are required to support successful brain circuitry and clinical gene therapy studies. However, converting large promoters into functional MiniPromoters, which can be used in vectors with limited capacity, remains challenging. In this study, we describe the generation of a novel version of alphaherpesvirus latency-associated promoter 2 (LAP2), which facilitates precise transgene expression exclusively in the neurons of the mouse brain while minimizing undesired targeting in peripheral tissues. Additionally, we aimed to create a compact neural promoter to facilitate packaging of larger transgenes. Our results revealed that MiniLAP2 (278 bp) drives potent transgene expression in all neurons in the mouse brain, with little to no expression in glial cells. In contrast to the native promoter, MiniLAP2 reduced tropism in the spinal cord and liver. No expression was detected in the kidney or skeletal muscle. In summary, we developed a minimal pan-neuronal promoter that drives specific and robust transgene expression in the mouse brain when delivered intravenously via AAV-PHP.eB vector. The use of this novel MiniPromoter may broaden the range of deliverable therapeutics and improve their safety and efficacy by minimizing the potential for off-target effects.
Somatic gene therapy will be one of the most exciting practices of genetic medicine in Africa and is primed to offer a "new life" for persons living with sickle cell disease (SCD). Recently, successful gene therapy trial...Somatic gene therapy will be one of the most exciting practices of genetic medicine in Africa and is primed to offer a "new life" for persons living with sickle cell disease (SCD). Recently, successful gene therapy trials for SCD in the USA have sparked a ray of hope within the SCD community in Africa. However, the high cost, estimated to exceed 1.5 million USD, continues to be a major concern for many stakeholders. While affordability is a key global health equity consideration, it is equally important to reflect on other ethical, legal and social issues (ELSIs) that may impact the responsible implementation of gene therapy for SCD in Africa. These include informed consent comprehension, risk of therapeutic misestimation and optimistic bias; priorities for SCD therapy trials; dearth of ethical and regulatory oversight for gene therapy in many African countries; identifying a favourable risk-benefit ratio; criteria for the selection of trial participants; decisional conflict in consent; standards of care; bounded justice; and genetic tourism. Given these ELSIs, we suggest that researchers, pharma, funders, global health agencies, ethics committees, science councils and SCD patient support/advocacy groups should work together to co-develop: (1) patient-centric governance for gene therapy in Africa, (2) public engagement and education materials, and (3) decision making toolkits for trial participants. It is also critical to establish harmonised ethical and regulatory frameworks for gene therapy in Africa, and for global health agencies to accelerate access to basic care for SCD in Africa, while simultaneously strengthening capacity for gene therapy.
Mateu-Bosch A, Segur-Bailach E, García-Villoria J
… +9 more, Gea-Sorlí S, Ruiz I, Del Rey J, Camps J, Guitart-Mampel M, Garrabou G, Tort F, Ribes A, Fillat C
Glutaric Aciduria type I (GA1) is a rare neurometabolic disorder caused by mutations in the GDCH gene encoding for glutaryl-CoA dehydrogenase (GCDH) in the catabolic pathway of lysine, hydroxylysine and tryptophan. GCDH...Glutaric Aciduria type I (GA1) is a rare neurometabolic disorder caused by mutations in the GDCH gene encoding for glutaryl-CoA dehydrogenase (GCDH) in the catabolic pathway of lysine, hydroxylysine and tryptophan. GCDH deficiency leads to increased concentrations of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body fluids and tissues. These metabolites are the main triggers of brain damage. Mechanistic studies supporting neurotoxicity in mouse models have been conducted. However, the different vulnerability to some stressors between mouse and human brain cells reveals the need to have a reliable human neuronal model to study GA1 pathogenesis. In the present work we generated a GCDH knockout (KO) in the human neuroblastoma cell line SH-SY5Y by CRISPR/Cas9 technology. SH-SY5Y-GCDH KO cells accumulate GA, 3-OHGA, and glutarylcarnitine when exposed to lysine overload. GA or lysine treatment triggered neuronal damage in GCDH deficient cells. SH-SY5Y-GCDH KO cells also displayed features of GA1 pathogenesis such as increased oxidative stress vulnerability. Restoration of the GCDH activity by gene replacement rescued neuronal alterations. Thus, our findings provide a human neuronal cellular model of GA1 to study this disease and show the potential of gene therapy to rescue GCDH deficiency.
Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the...Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K channels has been proposed as a treatment due to the outstanding ability of K channels to hyperpolarize neurons. However, the effects of K channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca-gated K channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.