Lauren Renner

Safety and Effects of the Vector for the Leber Hereditary Optic Neuropathy Gene Therapy Clinical Trial

IMPORTANCE We developed a novel strategy for treatment of Leber hereditary optic neuropathy (LHON) caused by a mutation in the nicotinamide adenine dinucleotide dehydrogenase subunit IV (ND4) mitochondrial gene. OBJECTIVE To demonstrate the safety and effects of the gene therapy vector to be used in a proposed gene therapy clinical trial. DESIGN AND SETTING In a series of laboratory experiments, we modified the mitochondrial ND4 subunit of complex I in the nuclear genetic code for import into mitochondria. The protein was targeted into the organelle by agency of a targeting sequence (allotopic expression). The gene was packaged into adeno-associated viral vectors and then vitreally injected into rodent, nonhuman primate, and ex vivo human eyes that underwent testing for expression and integration by immunohistochemical analysis and blue native polyacrylamide gel electrophoresis. During serial follow-up, the animal eyes underwent fundus photography, optical coherence tomography, and multifocal or pattern electroretinography. We tested for rescue of visual loss in rodent eyes also injected with a mutant G11778A ND4 homologue responsible for most cases of LHON. EXPOSURE Ocular infection with recombinant adeno-associated viral vectors containing a wild-type allotopic human ND4 gene. MAIN OUTCOMES AND MEASURES Expression of human ND4 and rescue of optic neuropathy induced by mutant human ND4. RESULTS We found human ND4 expressed in almost all mouse retinal ganglion cells by 1 week after injection and ND4 integrated into the mouse complex I. In rodent eyes also injected with a mutant allotopic ND4, wild-type allotopic ND4 prevented defective adenosine triphosphate synthesis, suppressed visual loss, reduced apoptosis of retinal ganglion cells, and prevented demise of axons in the optic nerve. Injection of ND4 in the ex vivo human eye resulted in expression in most retinal ganglion cells. Primates undergoing vitreal injection with the ND4 test article and followed up for 3 months had no serious adverse reactions. CONCLUSIONS AND RELEVANCE Expression of our allotopic ND4 vector in the ex vivo human eye, safety of the test article, rescue of the LHON mouse model, and the severe irreversible loss of visual function in LHON support clinical testing with mutated G11778A mitochondrial DNA in our patients.

LHON Gene Therapy Vector Prevents Visual Loss and Optic Neuropathy Induced by G11778A Mutant Mitochondrial DNA: Biodistribution and Toxicology Profile

PURPOSE To demonstrate safety and efficacy of allotopic human ND4 for treatment of a Lebers hereditary optic neuropathy (LHON) mouse model harboring the G11778A mitochondrial mutation. METHODS We induced LHON in mice by intravitreal injection of mutant (G11778A) human ND4 DNA, responsible for most cases of LHON, that was directed to mitochondria using an AAV2 vector to which we appended a mitochondrial targeting sequence to the VP2 capsid. We then attempted rescue of visual loss using our test article (ScAAV2-P1ND4v2) containing a synthetic nuclear encoded G11778G ND4 gene that was allotopically expressed. Control mice either were uninjected or received AAV2-GFP or AAV2-mCherry. We performed RT-PCR and confocal microscopy at 2 weeks post injection. Pattern electroretinograms (PERGs), spectral-domain optical coherence tomography (SD-OCT), histology, and transmission electron microscopy (TEM) were performed. For toxicology and biodistribution studies, the test article was administered intravitreally to rats and rhesus macaques at different doses. RESULTS Mutant and wild-type ND4 were efficiently expressed in the mitochondria of retinal ganglion cells (RGCs). Visual function assessed by serial PERGs and retinal structure by serial SD-OCT showed a significant rescue by the test article. Histology and ultrastructural analysis confirmed that loss of RGCs and demise of axons was prevented by ScAAV2-P1ND4v2. Rat and nonhuman primate biodistribution studies showed that vector spread outside the injected eye into spleen and lymph nodes was minimal. Histopathology of tissues and organs including the eyes was comparable to that of uninfected and saline-injected eyes. CONCLUSIONS Allotopically expressed wild-type ND4 prevents the phenotype induced by G11778A mitochondrial DNA with a toxicology profile acceptable for testing in a phase I clinical trial.

Effect of Ovarian Hormone Therapy on Cognition in the Aged Female Rhesus Macaque

Studies of the effect of hormone therapy on cognitive function in menopausal women have been equivocal, in part due to differences in the type and timing of hormone treatment. Here we cognitively tested aged female rhesus macaques on (1) the delayed response task of spatial working memory, (2) a visuospatial attention task that measured spatially and temporally cued reaction times, and (3) a simple reaction time task as a control for motor speed. After task acquisition, animals were ovariectomized (OVX). Their performance was compared with intact controls for 2 months, at which time no group differences were found. The OVX animals were then assigned to treatment with either a subcutaneous sham implant (OVX), 17-β estradiol (E) implant (OVX+E) or E implant plus cyclic oral progesterone (OVX+EP). All groups were then tested repeatedly over 12 months. The OVX+E animals performed significantly better on the delayed response task than all of the other groups for much of the 12 month testing period. The OVX+EP animals also showed improved performance in the delayed response task, but only at 30 s delays and with performance levels below that of OVX+E animals. The OVX+E animals also performed significantly better in the visuospatial attention task, particularly in the most challenging invalid cue condition; this difference also was maintained across the 12 month testing period. Simple reaction time was not affected by hormonal manipulation. These data demonstrate that chronic, continuous administration of E can exert multiple beneficial cognitive effects in aged, OVX rhesus macaque females. SIGNIFICANCE STATEMENT Hormone therapy after menopause is controversial. We tested the effects of hormone replacement in aged rhesus macaques, soon after surgically-induced menopause [ovariectomy (OVX)], on tests of memory and attention. Untreated ovarian-intact and OVX animals were compared with OVX animals receiving estradiol (E) alone or E with progesterone (P). E was administered in a continuous fashion via subcutaneous implant, whereas P was administered orally in a cyclic fashion. On both tests, E-treated animals performed better than the other 3 experimental groups across 1 year of treatment. Thus, in this monkey model, chronic E administered soon after the loss of ovarian hormones had long-term benefits for cognitive function.

Elevated Fundus Autofluorescence in Monkeys Deficient in Lutein, Zeaxanthin, and Omega-3 Fatty Acids.

Purpose We quantified fundus autofluorescence (FAF) in the nonhuman primate retina as a function of age and diets lacking lutein and zeaxanthin (L/Z) and omega-3 fatty acids. Methods Quantitative FAF was measured in a cross-sectional study of rhesus macaques fed a standard diet across the lifespan, and in aged rhesus macaques fed lifelong diets lacking L/Z and providing either adequate or deficient levels of omega-3 fatty acids. Macular FAF images were segmented into multiple regions of interest, and mean gray values for each region were calculated using ImageJ. The resulting FAF values were compared across ages within the standard diet animals, and among diet groups and regions. Results Fundus autofluorescence increased with age in the standard diet animals, and was highest in the perifovea. Monkeys fed L/Z-free diets with either adequate or deficient omega-3 fatty acids had significantly higher FAF overall than age-matched standard diet monkeys. Examined by region, those with adequate omega-3 fatty acids had higher FAF in the fovea and superior regions, while monkeys fed the diet lacking L/Z and omega-3 fatty acids had higher FAF in all regions. Conclusions Diets devoid of L/Z resulted in increased retinal autofluorescence, with the highest values in animals also lacking omega-3 fatty acids. The increase was equivalent to a 12- to 20-year acceleration in lipofuscin accumulation compared to animals fed a standard diet. Together these data add support for the role of these nutrients as important factors in lipofuscin accumulation, retinal aging, and progression of macular disease.

Changes in spontaneous activity assessed by accelerometry correlate with extent of cerebral ischemia-reperfusion injury in the nonhuman primate.

The use of accelerometry to monitor activity in human stroke patients has revealed strong correlations between objective activity measurements and subjective neurological findings. The goal of our study was to assess the applicability of accelerometry-based measurements in experimental animals undergoing surgically induced cerebral ischemia. Using a nonhuman primate cortical stroke model, we demonstrate for the first time that monitoring locomotor activity prior to and following cerebrovascular ischemic injury using an accelerometer is feasible in adult male rhesus macaques and that the measured activity outcomes significantly correlate with severity of brain injury. The use of accelerometry as an unobtrusive, objective preclinical efficacy determinant could complement standard practices involving subjective neurological scoring and magnetic resonance imaging in nonhuman primates. Similar activity monitoring devices to those employed in this study are currently in use in human clinical studies, underscoring the feasibility of this approach for assessing the clinical potential of novel treatments for cerebral ischemia.

Lutein is differentially deposited across brain regions following formula or breast feeding of infant rhesus macaques

Background Lutein, a yellow xanthophyll, selectively accumulates in primate retina and brain. Lutein may play a critical role in neural and retinal development, but few studies have investigated the impact of dietary source on its bioaccumulation in infants. Objective We explored the bioaccumulation of lutein in infant rhesus macaques following breastfeeding or formula-feeding. Methods From birth to 6 mo of age, male and female rhesus macaques (Macaca mulatta) were either breastfed (BF) (n = 8), fed a formula supplemented with lutein, zeaxanthin, β-carotene, and lycopene (237, 19.0, 74.2, and 338 nmol/kg, supplemented formula-fed; SF) (n = 8), or fed a formula with low amounts of these carotenoids (38.6, 2.3, 21.5, and 0 nmol/kg, unsupplemented formula-fed; UF) (n = 7). The concentrations of carotenoids in serum and tissues were analyzed by HPLC. Results At 6 mo of age, the BF group exhibited significantly higher lutein concentrations in serum, all brain regions, macular and peripheral retina, adipose tissue, liver, and other tissues compared to both formula-fed groups (P < 0.001). Lutein concentrations were higher in the SF group than in the UF group in serum and all tissues, with the exception of macular retina. Lutein was differentially distributed across brain areas, with the highest concentrations in the occipital cortex, regardless of the diet. Zeaxanthin was present in all brain regions but only in the BF infants; it was present in both retinal regions in all groups but was significantly enhanced in BF infants compared to either formula group (P < 0.001). β-Carotene accumulated across brain regions in all groups, but was not detected in retina. Although lycopene was found in many tissues of the SF group, it was not detected in the brain or retina. Conclusions Although carotenoid supplementation of infant formula significantly increased serum and tissue lutein concentrations compared to unsupplemented formula, concentrations were still well below those in BF infants. Regardless of diet, occipital cortex showed selectively higher lutein deposition than other brain regions, suggesting luteins role in visual processing in early life.

The effects of breastfeeding versus formula-feeding on cerebral cortex maturation in infant rhesus macaques

&NA; Breastfeeding is positively associated with several outcomes reflecting early brain development and cognitive functioning. Brain neuroimaging studies have shown that exclusively breastfed children have increased white matter and subcortical gray matter volume compared to formula‐fed children. However, it is difficult to disentangle the effects of nutrition in breast milk from other confounding factors that affect brain development, particularly in studies of human subjects. Among the nutrients provided by human breast milk are the carotenoid lutein and the natural form of tocopherol, both of which are selectively deposited in brain. Lutein is the predominant carotenoid in breast milk but not in most infant formulas, whereas infant formulas are supplemented with the synthetic form of tocopherol. In this study, a non‐human primate model was used to investigate the effects of breastfeeding versus formula‐feeding, as well as lutein and natural RRR‐&agr;‐tocopherol supplementation of infant formula, on brain maturation under controlled experimental conditions. Infant rhesus macaques (Macaca mulatta) were exclusively breastfed, or were fed infant formulas with different levels and sources of lutein and &agr;‐tocopherol. Of note, the breastfed group were mother‐reared whereas the formula‐fed infants were nursery‐reared. Brain structural and diffusion MR images were collected, and brain T2 was measured, at two, four and six months of age. The mother‐reared breastfed group was observed to differ from the formula‐fed groups by possessing higher diffusion fractional anisotropy (FA) in the corpus callosum, and lower FA in the cerebral cortex at four and six months of age. Cortical regions exhibiting the largest differences include primary motor, premotor, lateral prefrontal, and inferior temporal cortices. No differences were found between the formula groups. Although this study did not identify a nutritional component of breast milk that could be provided to infant formula to facilitate brain maturation consistent with that observed in breastfed animals, our findings indicate that breastfeeding promoted maturation of the corpus callosum and cerebral cortical gray matter in the absence of several confounding factors that affect studies in human infants. However, differences in rearing experience remain as a potential contributor to brain structural differences between breastfed and formula fed infants. HighlightsEffects of breastfeeding versus formula‐feeding, and versus lutein‐supplemented formula‐feeding, on brain maturation of infant non‐human primates are investigated by MRI.Breastfed infants exhibited greater maturity in corpus callosum compared to formulafed animals.Lower fractional anisotropy in cerebral cortical gray matter of breastfed compared to formula‐fed animals indicated more elaborate cortical differentiation due to breast feeding in infancy.Differences in brain maturation between lutein‐supplemented and unsupplemented formula‐feeding are not observed

Cell transplantation for retinal degeneration: Transition from rodent to nonhuman primate models

Transplantation of potentially therapeutic cells into the subretinal space is a promising prospective therapy for the treatment of retinal degenerative diseases including age-related macular degeneration (AMD). In rodent models with photoreceptor degeneration, subretinal transplantation of cell suspensions has repeatedly been demonstrated to rescue behaviorally measured vision, maintain electrophysiological responses from the retina and the brain, and slow the degeneration of rod and cone photoreceptors for extended periods. These studies have led to the initiation of a number of FDA-approved clinical trials for application of cell-based therapy for AMD and other retinal degenerative diseases. However, translation from rodent models directly into human clinical trials skips an important intermediary preclinical step that is needed to address critical issues for intraocular cell transplantation. These include determination of the most appropriate and least problematic surgical approach, the application of treatment in an eye with similar size and structure including the presence of a macula, and a thorough understanding of the immunological considerations regarding graft survival and the consequences of grafted cell rejection. This chapter will review these and related issues and will document current efforts to address these concerns.

Allogeneic iPSC-Derived RPE Cell Graft Failure Following Transplantation Into the Subretinal Space in Nonhuman Primates

Purpose To characterize the intraocular immune response following transplantation of iPS-derived allogeneic RPE cells into the subretinal space of non–immune-suppressed rhesus macaques. Methods GFP-labeled allogeneic iPS-derived RPE cells were transplanted into the subretinal space of one eye (n = 6), and into the contralateral eye 1 day to 4 weeks later, using a two-stage transretinal and transscleral approach. Retinas were examined pre- and post-surgery by color fundus photography, fundus autofluorescence, and optical coherence tomography (OCT) imaging. Animals were euthanized between 2 hours and 7 weeks following transplantation. T-cell (CD3), B-cell (CD20), and microglial (Iba1) responses were assessed immunohistochemically. Results Cells were delivered into the subretinal space in all eyes without leakage into the vitreous. Transplanted RPE cells were clearly visible at 4 days after surgery but were no longer detectable by 3 weeks. In localized areas within the bleb containing transplanted cells, T- and B-cell infiltrates and microglia were observed in the subretinal space and underlying choroid. A T-cell response predominated at 4 days, but converted to a B-cell response at 3 weeks. By 7 weeks, few infiltrates or microglia remained. Host RPE and choroid were disrupted in the immediate vicinity of the graft, with fibrosis in the subretinal space. Conclusions Engraftment of allogeneic RPE cells failed following transplantation into the subretinal space of rhesus macaques, likely due to rejection by the immune system. These data underscore the need for autologous cell sources and/or confirmation of adequate immune suppression to ensure survival of transplanted RPE cells.

New MiniPromoter Ple345 (NEFL) Drives Strong and Specific Expression in Retinal Ganglion Cells of Mouse and Primate Retina

Retinal gene therapy is leading the neurological gene therapy field, with 32 ongoing clinical trials of recombinant adeno-associated virus (rAAV)–based therapies. Importantly, over 50% of those trials are using restricted promoters from human genes. Promoters that restrict expression have demonstrated increased efficacy and can limit the therapeutic to the target cells thereby reducing unwanted off-target effects. Retinal ganglion cells are a critical target in ocular gene therapy; they are involved in common diseases such as glaucoma, rare diseases such as Lebers hereditary optic neuropathy, and in revolutionary optogenetic treatments. Here, we used computational biology and mined the human genome for the best genes from which to develop a novel minimal promoter element(s) designed for expression in restricted cell types (MiniPromoter) to improve the safety and efficacy of retinal ganglion cell gene therapy. Gene selection included the use of the first available droplet-based single-cell RNA sequencing (Drop-seq) dataset, and promoter design was bioinformatically driven and informed by a wide range of genomics datasets. We tested seven promoter designs from four genes in rAAV for specificity and quantified expression strength in retinal ganglion cells in mouse, and then the single best in nonhuman primate retina. Thus, we developed a new human-DNA MiniPromoter, Ple345 (NEFL), which in combination with intravitreal delivery in rAAV9 showed specific and robust expression in the retinal ganglion cells of the nonhuman-primate rhesus macaque retina. In mouse, we also developed MiniPromoters expressing in retinal ganglion cells, the hippocampus of the brain, a pan neuronal pattern in the brain, and peripheral nerves. As single-cell transcriptomics such as Drop-seq become available for other cell types, many new opportunities for additional novel restricted MiniPromoters will present.