ENGINEERED NANOBODIES WITH PROGRAMMABLE TARGET ANTIGEN PROTEOLYSIS (PTAP) FUSIONS REGULATE INTRACELLULAR ALPHA-SYNUCLEIN IN VITRO AND IN VIVO.

Alpha-synuclein (αSyn) aggregation and the formation of Lewy pathology (LP) is a foundational pathophysiological phenomenon in synucleinopathies. Delivering therapeutic single-chain and single-domain antibodies that bind pathogenic targets can disrupt intracellular aggregation. The fusion of antibody fragments to a negatively-charged proteasomal targeting motif (PEST) creates bifunctional constructs that enhance both solubility and turnover. With sequence-specific point mutations of PEST sequences that modulate proteasomal degradation efficiency, we report the creation of Programmable Target Antigen Proteolysis (PTAP) technology that can provide graded control over the levels of target antigens. We have previously demonstrated our lead anti-αSyn intrabody, VH14-PEST, is capable of reducing the pathological burden of synucleinopathy in vitro and in vivo. Here, we report a family of fully humanized VH14-PTAP constructs for controllable, therapeutic targeting of intracellular α-Syn. In cells, we demonstrate successful target engagement and efficacy of VH14-hPEST intrabodies, and validate proof-of-principle in human cells using 3D human organoids derived from PD-patient induced pluripotent stem cells (iPSC). In two synuclein-based rat models, PTAP intrabodies attenuated nigral αSyn pathology, preserved nigrostriatal dopaminergic tone, and slowed the propagation of αSyn pathology. These data demonstrate the potency of intracellular αSyn targeting as a method to alleviate pathology and highlight the potential clinical utility of PTAP intrabodies.

Optimizing maturity and dose of iPSC-derived dopamine progenitor cell therapy for Parkinson's disease.

In pursuit of treating Parkinson's disease with cell replacement therapy, differentiated induced pluripotent stem cells (iPSC) are an ideal source of midbrain dopaminergic (mDA) cells. We previously established a protocol for differentiating iPSC-derived post-mitotic mDA neurons capable of reversing 6-hydroxydopamine-induced hemiparkinsonism in rats. In the present study, we transitioned the iPSC starting material and defined an adapted differentiation protocol for further translation into a clinical cell transplantation therapy. We examined the effects of cellular maturity on survival and efficacy of the transplants by engrafting mDA progenitors (cryopreserved at 17 days of differentiation, D17), immature neurons (D24), and post-mitotic neurons (D37) into immunocompromised hemiparkinsonian rats. We found that D17 progenitors were markedly superior to immature D24 or mature D37 neurons in terms of survival, fiber outgrowth and effects on motor deficits. Intranigral engraftment to the ventral midbrain demonstrated that D17 cells had a greater capacity than D24 cells to innervate over long distance to forebrain structures, including the striatum. When D17 cells were assessed across a wide dose range (7,500-450,000 injected cells per striatum), there was a clear dose response with regards to numbers of surviving neurons, innervation, and functional recovery. Importantly, although these grafts were derived from iPSCs, we did not observe teratoma formation or significant outgrowth of other cells in any animal. These data support the concept that human iPSC-derived D17 mDA progenitors are suitable for clinical development with the aim of transplantation trials in patients with Parkinson's disease.

Enhanced CNS transduction from AAV.PHP.eB infusion into the cisterna magna of older adult rats compared to AAV9.

The development of high efficiency, central nervous system (CNS) targeting AAV-based gene therapies is necessary to address challenges in both pre-clinical and clinical investigations. The engineered capsids, AAV.PHP.B and AAV.PHP.eB, show vastly improved blood-brain barrier penetration compared to their parent serotype, AAV9, but with variable effect depending on animal system, strain, and delivery route. As most characterizations of AAV.PHP variants have been performed in mice, it is currently unknown whether AAV.PHP variants improve CNS targeting when delivered intrathecally in rats. We evaluated the comparative transduction efficiencies of equititer doses (6 × 1011vg) of AAV.PHP.eB-CAG-GFP and AAV9-CAG-GFP when delivered into the cisterna magna of 6-9-month old rats. Using both quantitative and qualitative assessments, we observed consistently superior biodistribution of GFP+ cells and fibers in animals treated with AAV.PHP.eB compared to those treated with AAV9. Enhanced GFP signal was uniformly observed throughout rostrocaudal brain regions in AAV.PHP.eB-treated animals with matching GFP protein expression detected in the forebrain, midbrain, and cerebellum. Collectively, these data illustrate the benefit of intracisternal infusions of AAV.PHP.eB as an optimal system to distribute CNS gene therapies in preclinical investigations of rats, and may have important translational implications for the clinical CNS targeting.

A historical review of multiple system atrophy with a critical appraisal of cellular and animal models.

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA), and dysautonomia with cerebellar ataxia or parkinsonian motor features. Isolated autonomic dysfunction with predominant genitourinary dysfunction and orthostatic hypotension and REM sleep behavior disorder are common characteristics of a prodromal phase, which may occur years prior to motor-symptom onset. MSA is a unique synucleinopathy, in which alpha-synuclein (aSyn) accumulates and forms insoluble inclusions in the cytoplasm of oligodendrocytes, termed glial cytoplasmic inclusions (GCIs). The origin of, and precise mechanism by which aSyn accumulates in MSA are unknown, and, therefore, disease-modifying therapies to halt or slow the progression of MSA are currently unavailable. For these reasons, much focus in the field is concerned with deciphering the complex neuropathological mechanisms by which MSA begins and progresses through the course of the disease. This review focuses on the history, etiopathogenesis, neuropathology, as well as cell and animal models of MSA.

Gene Therapy to Modulate Alpha-Synuclein in Synucleinopathies.

The protein alpha-Synuclein (α-Syn) is a key contributor to the etiology of Parkinson's disease (PD) with aggregation, trans-neuronal spread, and/or depletion of α-Syn being viewed as crucial events in the molecular processes that result in neurodegeneration. The exact succession of pathological occurrences that lead to neuronal death are still largely unknown and are likely to be multifactorial in nature. Despite this unknown, α-Syn dose and stability, autophagy-lysosomal dysfunction, and inflammation, amongst other cellular impairments, have all been described as participatory events in the neurodegenerative process. To that end, in this review we discuss the logical points for gene therapy to intervene in α-Syn-mediated disease and review the preclinical body of work where gene therapy has been used, or could conceptually be used, to ameliorate α-Syn induced neurotoxicity. We discuss gene therapy in the traditional sense of modulating gene expression, as well as the use of viral vectors and nanoparticles as methods to deliver other therapeutic modalities.

Mitomycin-C treatment during differentiation of induced pluripotent stem cell-derived dopamine neurons reduces proliferation without compromising survival or function in vivo.

Nongenetic methodologies to reduce undesirable proliferation would be valuable when generating dopamine neurons from stem cells for transplantation in Parkinson's disease (PD). To this end, we modified an established method for controlled differentiation of human induced pluripotent stem cells (iPSCs) into midbrain dopamine neurons using two distinct methods: omission of FGF8 or the in-process use of the DNA cross-linker mitomycin-C (MMC). We transplanted the cells to athymic rats with unilateral 6-hydroxydopamine lesions and monitored long-term survival and function of the grafts. Transplants of cells manufactured using MMC had low proliferation while still permitting robust survival and function comparable to that seen with transplanted dopamine neurons derived using genetic drug selection. Conversely, cells manufactured without FGF8 survived transplantation but exhibited poor in vivo function. Our results suggest that MMC can be used to reduce the number of proliferative cells in stem cell-derived postmitotic neuron preparations for use in PD cell therapy.

Viral-based rodent and nonhuman primate models of multiple system atrophy: Fidelity to the human disease.

Multiple system atrophy (MSA) is a rare and extremely debilitating progressive neurodegenerative disease characterized by variable combinations of parkinsonism, cerebellar ataxia, dysautonomia, and pyramidal dysfunction. MSA is a unique synucleinopathy, in which alpha synuclein-rich aggregates are present in the cytoplasm of oligodendroglia. The precise origin of the alpha synuclein (aSyn) found in the glial cytoplasmic inclusions (GCIs) as well the mechanisms of neurodegeneration in MSA remain unclear. Despite this fact, cell and animal models of MSA rely on oligodendroglial overexpression of aSyn. In the present study, we utilized a novel oligotrophic AAV, Olig001, to overexpress aSyn specifically in striatal oligodendrocytes of rats and nonhuman primates in an effort to further characterize our novel viral vector-mediated MSA animal models. Using two cohorts of animals with 10-fold differences in Olig001 vector titers, we show a dose-dependent formation of MSA-like pathology in rats. High titer of Olig001-aSyn in these animals were required to produce the formation of pS129+ and proteinase K resistant aSyn-rich GCIs, demyelination, and neurodegeneration. Using this knowledge, we injected high titer Olig001 in the putamen of cynomolgus macaques. After six months, histological analysis showed that oligodendroglial overexpression of aSyn resulted in the formation of hallmark GCIs throughout the putamen, demyelination, a 44% reduction of striatal neurons and a 12% loss of nigral neurons. Furthermore, a robust inflammatory response similar to MSA was produced in Olig001-aSyn NHPs, including microglial activation, astrogliosis, and a robust infiltration of T cells into the CNS. Taken together, oligodendroglial-specific viral vector-mediated overexpression of aSyn in rats and nonhuman primates faithfully reproduces many of the pathological disease hallmarks found in MSA. Future studies utilizing these large animal models of MSA would prove extremely valuable as a pre-clinical platform to test novel therapeutics that are so desperately needed for MSA.

T cell infiltration in both human multiple system atrophy and a novel mouse model of the disease.

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by abnormal accumulation of alpha-synuclein (α-syn) in oligodendrocytes accompanied by inflammation, demyelination, and subsequent synapse and neuronal loss. Little is known about the mechanisms of neurodegeneration in MSA. However, recent work has highlighted the important role of the immune system to the pathophysiology of other synuclein-related diseases such as Parkinson's disease. In this study, we investigated postmortem brain tissue from MSA patients and control subjects for evidence of immune activation in the brain. We found a significant increase of HLA-DR+ microglia in the putamen and substantia nigra of MSA patient tissue compared to controls, as well as significant increases in CD3+, CD4+, and CD8+ T cells in these same brain regions. To model MSA in vivo, we utilized a viral vector that selectively overexpresses α-syn in oligodendrocytes (Olig001-SYN) with > 95% tropism in the dorsal striatum of mice, resulting in demyelination and neuroinflammation similar to that observed in human MSA. Oligodendrocyte transduction with this vector resulted in a robust inflammatory response, which included increased MHCII expression on central nervous system (CNS) resident microglia, and infiltration of pro-inflammatory monocytes into the CNS. We also observed robust infiltration of CD4 T cells into the CNS and antigen-experienced CD4 T cells in the draining cervical lymph nodes. Importantly, genetic deletion of TCR-ß or CD4 T cells attenuated α-syn-induced inflammation and demyelination in vivo. These results suggest that T cell priming and infiltration into the CNS are key mechanisms of disease pathogenesis in MSA, and therapeutics targeting T cells may be disease modifying.

α-Synuclein nonhuman primate models of Parkinson's disease.

Proper understanding of the mechanism(s) by which α-synuclein misfolds and propagates may hold the key to unraveling the complex pathophysiology of Parkinson's disease. A more complete understanding of the disease itself, as well as establishing animal models that fully recapitulate pathological and functional disease progression, are needed to develop treatments that will delay, halt or reverse the disease course. Traditional neurotoxin-based animal models fail to mimic crucial aspects of Parkinson's and thus are not relevant for the study of neuroprotection and disease-modifying therapies. Therefore, a new era of animal models centered on α-synuclein has emerged with the utility of nonhuman primates in these studies beginning to become important. Indeed, disease modeling in nonhuman primates offers a more similar anatomical and genetic background to humans, and the ability to assess complex behavioral impairments that are difficult to test in rodents. Furthermore, results obtained from monkey studies translate better to applications in humans. In this review, we highlight the importance of α-synuclein in Parkinson's disease and discuss the development of α-synuclein based nonhuman primate models.

Novel oligodendroglial alpha synuclein viral vector models of multiple system atrophy: studies in rodents and nonhuman primates.

Multiple system atrophy (MSA) is a horrible and unrelenting neurodegenerative disorder with an uncertain etiology and pathophysiology. MSA is a unique proteinopathy in which alpha-synuclein (α-syn) accumulates preferentially in oligodendroglia rather than neurons. Glial cytoplasmic inclusions (GCIs) of α-syn are thought to elicit changes in oligodendrocyte function, such as reduced neurotrophic support and demyelination, leading to neurodegeneration. To date, only a murine model using one of three promoters exist to study this disease. We sought to develop novel rat and nonhuman primate (NHP) models of MSA by overexpressing α-syn in oligodendroglia using a novel oligotrophic adeno-associated virus (AAV) vector, Olig001. To establish tropism, rats received intrastriatal injections of Olig001 expressing GFP. Histological analysis showed widespread expression of GFP throughout the striatum and corpus callosum with >95% of GFP+ cells co-localizing with oligodendroglia and little to no expression in neurons or astrocytes. We next tested the efficacy of this vector in rhesus macaques with intrastriatal injections of Olig001 expressing GFP. As in rats, we observed a large number of GFP+ cells in gray matter and white matter tracts of the striatum and the corpus callosum, with 90-94% of GFP+ cells co-localizing with an oligodendroglial marker. To evaluate the potential of our vector to elicit MSA-like pathology in NHPs, we injected rhesus macaques intrastriatally with Olig001 expressing the α-syn transgene. Histological analysis 3-months after injection demonstrated widespread α-syn expression throughout the striatum as determined by LB509 and phosphorylated serine-129 α-syn immunoreactivity, all of which displayed as tropism similar to that seen with GFP. As in MSA, Olig001-α-syn GCIs in our model were resistant to proteinase K digestion and caused microglial activation. Critically, demyelination was observed in the white matter tracts of the corpus callosum and striatum of Olig001-α-syn but not Olig001-GFP injected animals, similar to the human disease. These data support the concept that this vector can provide novel rodent and nonhuman primate models of MSA.

Cryopreservation Maintains Functionality of Human iPSC Dopamine Neurons and Rescues Parkinsonian Phenotypes In Vivo.

A major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD) is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle, midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA) and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic iPSC-mDA neurons retained high viability with gene, protein, and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth, supporting the translational development of pluripotent cell-based therapies in PD.

Robust graft survival and normalized dopaminergic innervation do not obligate recovery in a Parkinson disease patient.

OBJECTIVE: The main goal of dopamine cell replacement therapy in Parkinson disease (PD) is to provide clinical benefit mediated by graft survival with nigrostriatal reinnervation. We report a dichotomy between graft structure and clinical function in a patient dying 16 years following fetal nigral grafting. METHODS: A 55-year-old levodopa-responsive woman with PD received bilateral putaminal fetal mesencephalic grafts as part of an NIH-sponsored double-blind sham-controlled trial. The patient never experienced clinical benefit, and her course was complicated by the development of graft-related dyskinesias. Fluorodopa positron emission tomography demonstrated significant increases postgrafting bilaterally. She experienced worsening of parkinsonism with severe dyskinesias, and underwent subthalamic nucleus deep brain stimulation 8 years after grafting. She died 16 years after transplantation. RESULTS: Postmortem analyses confirmed the diagnosis of PD and demonstrated >300,000 tyrosine hydroxylase (TH)-positive grafted cells per side with normalized striatal TH-immunoreactive fiber innervation and bidirectional synaptic connectivity. Twenty-seven percent and 17% of grafted neurons were serine 129-phosphorylated α-synuclein positive in the left and right putamen, respectively. INTERPRETATION: These findings represent the largest number of surviving dopamine neurons and the densest and most widespread graft-mediated striatal dopamine reinnervation following a transplant procedure reported to date. Despite this, clinical recovery was not observed. Furthermore, the grafts were associated with a form of dyskinesias that resembled diphasic dyskinesia and persisted in the off-medication state. We hypothesize that the grafted cells produced a low level of dopamine sufficient to cause a levodopa-independent continuous form of diphasic dyskinesias, but insufficient to provide an antiparkinsonian benefit. ANN NEUROL 2017;81:46-57.