Engineering an Autonucleolytic Mammalian Suspension Host Cell Line to Reduce DNA Impurity Levels in Serum-Free Lentiviral Process Streams.

We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.

Fetal gene therapy.

Fetal gene therapy was first proposed toward the end of the 1990s when the field of gene therapy was, to quote the Gartner hype cycle, at its "peak of inflated expectations." Gene therapy was still an immature field but over the ensuing decade, it matured and is now a clinical and market reality. The trajectory of treatment for several genetic diseases is toward earlier intervention. The ability, capacity, and the will to diagnose genetic disease early-in utero-improves day by day. A confluence of clinical trials now signposts a trajectory toward fetal gene therapy. In this review, we recount the history of fetal gene therapy in the context of the broader field, discuss advances in fetal surgery and diagnosis, and explore the full ambit of preclinical gene therapy for inherited metabolic disease.

Advanced Formulation Approaches for Emerging Therapeutic Technologies.

In addition to proteins, discussed in the Chapter "Advances in Vaccine Adjuvants: Nanomaterials and Small Molecules", there are a wide range of alternatives to small molecule active ingredients. Cells, extracellular vesicles, and nucleic acids in particular have attracted increasing research attention in recent years. There are now a number of products on the market based on these emerging technologies, the most famous of which are the mRNA-based vaccines against SARS-COV-2. These advanced therapeutic moieties are challenging to formulate however, and there remain significant challenges for their more widespread use. In this chapter, we consider the potential and bottlenecks for developing further medical products based on these systems. Cells, extracellular vesicles, and nucleic acids will be discussed in terms of their mechanism of action, the key requirements for translation, and how advanced formulation approaches can aid their future development. These points will be presented with selected examples from the literature, and with a focus on the formulations which have made the transition to clinical trials and clinical products.

Microfluidic production of nanogels as alternative triple transfection reagents for the manufacture of adeno-associated virus vectors.

Adeno-associated viral vectors (AAVs) have proved a mainstay in gene therapy, owing to their remarkable transduction efficiency and safety profile. Their production, however, remains challenging in terms of yield, the cost-effectiveness of manufacturing procedures and large-scale production. In this work, we present nanogels produced by microfluidics as a novel alternative to standard transfection reagents such as polyethylenimine-MAX (PEI-MAX) for the production of AAV vectors with comparable yields. Nanogels were formed at pDNA weight ratios of 1 : 1 : 2 and 1 : 1 : 3, of pAAV cis-plasmid, pDG9 capsid trans-plasmid and pHGTI helper plasmid respectively, where vector yields at a small scale showed no significant difference to those of PEI-MAX. Weight ratios of 1 : 1 : 2 showed overall higher titers than 1 : 1 : 3, where nanogels with nitrogen/phosphate ratios of 5 and 10 produced yields of ≈8.8 × 108 vg mL-1 and ≈8.1 × 108 vg mL-1 respectively compared to ≈1.1 × 109 vg mL-1 for PEI-MAX. In larger scale production, optimised nanogels produced AAV at a titer of ≈7.4 × 1011 vg mL-1, showing no statistical difference from that of PEI-MAX at ≈1.2 × 1012 vg mL-1, indicating that equivalent titers can be achieved with easy-to-implement microfluidic technology at comparably lower costs than traditional reagents.

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-µ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

Gene Therapy for Lysosomal Storage Disorders: Ongoing Studies and Clinical Development.

Rare monogenic disorders such as lysosomal diseases have been at the forefront in the development of novel treatments where therapeutic options are either limited or unavailable. The increasing number of successful pre-clinical and clinical studies in the last decade demonstrates that gene therapy represents a feasible option to address the unmet medical need of these patients. This article provides a comprehensive overview of the current state of the field, reviewing the most used viral gene delivery vectors in the context of lysosomal storage disorders, a selection of relevant pre-clinical studies and ongoing clinical trials within recent years.

Clinical and Molecular Features of Early Infantile Niemann Pick Type C Disease.

Niemann Pick disease type C (NPC) is a neurovisceral disorder due to mutations in NPC1 or NPC2. This review focuses on poorly characterized clinical and molecular features of early infantile form of NPC (EIF) and identified 89 cases caused by NPC1 (NPC1) and 16 by NPC2 (NPC2) mutations. Extra-neuronal features were common; visceromegaly reported in 80/89 NPC1 and in 15/16 NPC2, prolonged jaundice in 30/89 NPC1 and 7/16 NPC2. Early lung involvement was present in 12/16 NPC2 cases. Median age of neurological onset was 12 (0-24) and 7.5 (0-24) months in NPC1 and NPC2 groups, respectively. Developmental delay and hypotonia were the commonest first detected neurological symptoms reported in 39/89 and 18/89 NPC1, and in 8/16 and 10/16 NPC2, respectively. Additional neurological symptoms included vertical supranuclear gaze palsy, dysarthria, cataplexy, dysphagia, seizures, dystonia, and spasticity. The following mutations in homozygous state conferred EIF: deletion of exon 1+promoter, c.3578_3591 + 9del, c.385delT, p.C63fsX75, IVS21-2delATGC, c. 2740T>A (p.C914S), c.3584G>T (p.G1195V), c.3478-6T>A, c.960_961dup (p.A321Gfs*16) in NPC1 and c.434T>A (p.V145E), c.199T>C (p.S67P), c.133C>T (p.Q45X), c.141C>A (p.C47X) in NPC2. This comprehensive analysis of the EIF type of NPC will benefit clinical patient management, genetic counselling, and assist design of novel therapy trials.

Neuroprotective Effects of Diabetes Drugs for the Treatment of Neonatal Hypoxia-Ischemia Encephalopathy.

The perinatal period represents a time of great vulnerability for the developing brain. A variety of injuries can result in death or devastating injury causing profound neurocognitive deficits. Hypoxic-ischemic neonatal encephalopathy (HIE) remains the leading cause of brain injury in term infants during the perinatal period with limited options available to aid in recovery. It can result in long-term devastating consequences with neurologic complications varying from mild behavioral deficits to severe seizure, intellectual disability, and/or cerebral palsy in the newborn. Despite medical advances, the only viable option is therapeutic hypothermia which is classified as the gold standard but is not used, or may not be as effective in preterm cases, infection-associated cases or low resource settings. Therefore, alternatives or adjunct therapies are urgently needed. Ongoing research continues to advance our understanding of the mechanisms contributing to perinatal brain injury and identify new targets and treatments. Drugs used for the treatment of patients with type 2 diabetes mellitus (T2DM) have demonstrated neuroprotective properties and therapeutic efficacy from neurological sequelae following HIE insults in preclinical models, both alone, or in combination with induced hypothermia. In this short review, we have focused on recent findings on the use of diabetes drugs that provide a neuroprotective effect using in vitro and in vivo models of HIE that could be considered for clinical translation as a promising treatment.

Experimental gene therapies for the NCLs.

The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of rare monogenic neurodegenerative diseases predominantly affecting children. All NCLs are lethal and incurable and only one has an approved treatment available. To date, 13 NCL subtypes (CLN1-8, CLN10-14) have been identified, based on the particular disease-causing defective gene. The exact functions of NCL proteins and the pathological mechanisms underlying the diseases are still unclear. However, gene therapy has emerged as an attractive therapeutic strategy for this group of conditions. Here we provide a short review discussing updates on the current gene therapy studies for the NCLs.

Correction: Impaired cellular bioenergetics caused by GBA1 depletion sensitizes neurons to calcium overload.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Systemic AAV9 gene therapy using the synapsin I promoter rescues a mouse model of neuronopathic Gaucher disease but with limited cross-correction potential to astrocytes.

Gaucher disease is caused by mutations in the GBA gene, which encodes for the lysosomal enzyme ß-glucocerebrosidase (GCase), resulting in the accumulation of storage material in visceral organs and in some cases the brain of affected patients. While there is a commercially available treatment for the systemic manifestations, neuropathology still remains untreatable. We previously demonstrated that gene therapy represents a feasible therapeutic tool for the treatment of the neuronopathic forms of Gaucher disease (nGD). In order to further enhance the therapeutic affects to the central nervous system, we systemically delivered an adeno-associated virus (AAV) serotype 9 carrying the human GBA gene under control of a neuron-specific promoter to an nGD mouse model. Gene therapy increased the life span of treated animals, rescued the lethal neurodegeneration, normalized the locomotor behavioural defects and ameliorated the visceral pathology. Together, these results provided further indication of gene therapy as a possible effective treatment option for the neuropathic forms of Gaucher disease.

Impaired cellular bioenergetics caused by GBA1 depletion sensitizes neurons to calcium overload.

Heterozygous mutations of the lysosomal enzyme glucocerebrosidase (GBA1) represent the major genetic risk for Parkinson's disease (PD), while homozygous GBA1 mutations cause Gaucher disease, a lysosomal storage disorder, which may involve severe neurodegeneration. We have previously demonstrated impaired autophagy and proteasomal degradation pathways and mitochondrial dysfunction in neurons from GBA1 knockout (gba1-/-) mice. We now show that stimulation with physiological glutamate concentrations causes pathological [Ca2+]c responses and delayed calcium deregulation, collapse of mitochondrial membrane potential and an irreversible fall in the ATP/ADP ratio. Mitochondrial Ca2+ uptake was reduced in gba1-/- cells as was expression of the mitochondrial calcium uniporter. The rate of free radical generation was increased in gba1-/- neurons. Behavior of gba1+/- neurons was similar to gba1-/- in terms of all variables, consistent with a contribution of these mechanisms to the pathogenesis of PD. These data signpost reduced bioenergetic capacity and [Ca2+]c dysregulation as mechanisms driving neurodegeneration.

Neonatal brain-directed gene therapy rescues a mouse model of neurodegenerative CLN6 Batten disease.

The neuronal ceroid lipofuscinoses (NCLs), more commonly referred to as Batten disease, are a group of inherited lysosomal storage disorders that present with neurodegeneration, loss of vision and premature death. There are at least 13 genetically distinct forms of NCL. Enzyme replacement therapies and pre-clinical studies on gene supplementation have shown promising results for NCLs caused by lysosomal enzyme deficiencies. The development of gene therapies targeting the brain for NCLs caused by defects in transmembrane proteins has been more challenging and only limited therapeutic effects in animal models have been achieved so far. Here, we describe the development of an adeno-associated virus (AAV)-mediated gene therapy to treat the neurodegeneration in a mouse model of CLN6 disease, a form of NCL with a deficiency in the membrane-bound protein CLN6. We show that neonatal bilateral intracerebroventricular injections with AAV9 carrying CLN6 increase lifespan by more than 90%, maintain motor skills and motor coordination and reduce neuropathological hallmarks of Cln6-deficient mice up to 23 months post vector administration. These data demonstrate that brain-directed gene therapy is a valid strategy to treat the neurodegeneration of CLN6 disease and may be applied to other forms of NCL caused by transmembrane protein deficiencies in the future.

Inhibition of Mitochondrial Complex I Impairs Release of α-Galactosidase by Jurkat Cells.

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.

Epilepsy Gene Therapy Using an Engineered Potassium Channel.

Refractory focal epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon optimized for human expression and mutated to accelerate the recovery of the channels from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a nonintegrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled preclinical trial, the EKC lentivector robustly reduced seizure frequency in a male rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. When packaged into an adeno-associated viral vector (AAV2/9), the EKC gene was also effective at suppressing seizures in a male rat model of temporal lobe epilepsy. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as being ready for clinical translation in the treatment of refractory focal epilepsy.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects up to 0.3% of the population. Although epilepsy surgery can be effective, it is limited by risks to normal brain function. We have developed a gene therapy that builds on a mechanistic understanding of altered neuronal and circuit excitability in cortical epilepsy. The potassium channel gene KCNA1 was mutated to bypass post-transcriptional editing and was packaged in a nonintegrating lentivector to reduce the risk of insertional mutagenesis. A randomized, blinded preclinical study demonstrated therapeutic effectiveness in a rodent model of focal neocortical epilepsy. Adeno-associated viral delivery of the channel to both hippocampi was also effective in a model of temporal lobe epilepsy. These results support clinical translation to address a major unmet need.

Clinical challenges and future therapeutic approaches for neuronal ceroid lipofuscinosis.

Treatment of the neuronal ceroid lipofuscinoses, also known as Batten disease, is at the start of a new era because of diagnostic and therapeutic advances relevant to this group of inherited neurodegenerative and life-limiting disorders that affect children. Diagnosis has improved with the use of comprehensive DNA-based tests that simultaneously screen for many genes. The identification of disease-causing mutations in 13 genes provides a basis for understanding the molecular mechanisms underlying neuronal ceroid lipofuscinoses, and for the development of targeted therapies. These targeted therapies include enzyme replacement therapies, gene therapies targeting the brain and the eye, cell therapies, and pharmacological drugs that could modulate defective molecular pathways. Such therapeutic developments have the potential to enable earlier diagnosis and better targeted therapeutic management. The first approved treatment is an intracerebroventricularly administered enzyme for neuronal ceroid lipofuscinosis type 2 disease that delays symptom progression. Efforts are underway to make similar progress for other forms of the disorder.