Alistair Wilson

A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Bioactivity of AAV2-neurturin gene therapy (CERE-120): Differences between Parkinson's disease and nonhuman primate brains†

Background: AAV2‐neurturin (CERE‐120) is designed to deliver the neurotrophic‐factor, neurturin, to the striatum to restore and protect degenerating nigrostriatal neurons in Parkinsons disease (PD). A common hypothesis is that following expression in the striatum, neurotrophic‐factors like neurturin (NRTN) will be transported from degenerating terminals to their cell bodies in the substantia nigra pars compacta (SNc). Methods: We tested this concept using immunohistochemistry, comparing the bioactivity of AAV2‐neurturin in brains of PD patients versus those of nonhuman primates similarly treated. Results: NRTN‐immunostaining in the targeted striatum was seen in all PD cases (mean putaminal coverage: ∼15% by volume); comparable expression was observed in young, aged, and parkinsonian monkeys. In the SNc cell bodies, however, only rare evidence of neurturin was seen in PD, while ample evidence of intense nigral‐NRTN was observed in all monkeys. NRTN‐expression was associated with occasional, sparse TH‐induction in the striatum of PD, but nothing apparent in the SNc. In primates, NRTN produced robust TH‐induction throughout the nigrostriatal neurons. Discussion: These data provide the first evidence that gene therapy can increase expression of a neurotrophic‐factor deep in the PD brain and that clear but modest enhancement of degenerating neurons can be induced. They also provide important insight regarding deficiencies in the status of nigrostriatal neurons in advanced PD, suggesting that serious axon‐transport deficits reduced the bioactivity of AAV2‐NRTN by limiting the protein exposed to the cell body. Thus, future efforts using neurotrophic‐factors to treat neurodegenerative diseases will need to target both the terminal fields and the cell bodies of degenerating neurons to assure maximal benefit is achieved.

AAV2-mediated delivery of human neurturin to the rat nigrostriatal system: Long-term efficacy and tolerability of CERE-120 for Parkinson's disease

Neurturin (NTN) is a neurotrophic factor with known potential to protect and restore the function of dopaminergic substantia nigra neurons whose degeneration has been most closely linked to the major motor deficits in Parkinsons disease (PD). CERE-120, an adeno-associated virus serotype 2 (AAV2)-based gene delivery vector encoding human NTN, is being developed as a potential therapeutic for PD. In a series of preclinical studies reported herein, CERE-120 delivery to the striatum produced a dose-related neuroprotection of nigrostriatal neurons in the rat 6-hydroxydopamine (6-OHDA) lesion model. Long-lasting efficacy of CERE-120 was evidenced by substantia nigra cell protection, preserved fiber innervation of the striatum, and behavioral recovery for at least 6 months. In addition, striatal infusion of CERE-120 was found to have a safety and tolerability profile devoid of side effects or toxicological responses, for at least 12 months post-treatment, even at dose multiples 125 times that of the lowest efficacious dose tested. These results support the ongoing CERE-120 clinical program in PD patients.

Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo

The PEC‐01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC‐01 candidate product) and transplanted into mice, can mature into glucose‐responsive insulin‐secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC‐01 cells such that 73%–80% of the cell population consisted of PDX1‐positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet‐like cells (ICs) that reproducibly contained 73%–89% endocrine cells, of which approximately 40%–50% expressed insulin. A large fraction of these insulin‐positive cells were single hormone‐positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%–98% endocrine cells and 1%–3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC‐01 candidate product, demonstrating conclusively that in vitro‐produced hESC‐derived insulin‐producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important β cell genes, the cells contained relatively low levels of several maturity‐associated markers. Correlating with this, the time to function of ICs was similar to PEC‐01 cells, indicating that ICs required cell‐autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host.

Expression, bioactivity, and safety 1 year after adeno-associated viral vector type 2-mediated delivery of neurturin to the monkey nigrostriatal system support cere-120 for Parkinson's disease.

OBJECTIVEParkinsons disease is characterized by profound motor deficits that result mainly as a consequence of degeneration of midbrain dopaminergic neurons. No current therapy slows or halts disease progression. Neurturin (NTN) and glial cell line–derived neurotrophic factor have potent neuroprotective and neurorestorative effects on dopaminergic neurons, but their use in treating Parkinsons disease has been limited by significant delivery obstacles. In this study, we examined the long-term expression, bioactivity, and safety/tolerability of CERE-120, an adeno-associated virus type 2 vector encoding human NTN, after bilateral stereotactic delivery to the striatum of nonhuman primates. METHODSTwelve naïve rhesus macaques received bilateral stereotactic injections of 1 of 2 CERE-120 doses or vehicle to the caudate and putamen. Neurological and clinical parameters were monitored for up to 1 year postadministration, after which animals were sacrificed for histological analyses. RESULTSDose-related NTN expression was observed at 1 year and was associated with enhanced tyrosine hydroxylase immunolabeling in the striatum, hypertrophy of tyrosine hydroxylase–positive cells in the substantia nigra, and induction of extracellular signal–regulated kinase signaling in the substantia nigra. Extensive, formal analyses, conducted in accordance with Good Laboratory Practice Regulations, across multiple time points revealed no evidence of clinical, neurological, or systemic toxicity. CONCLUSIONThe present study provides evidence of long-term expression and bioactivity of NTN on the dopaminergic nigrostriatal system after bilateral stereotactic delivery of CERE-120 to the striatum. Furthermore, no evidence of any adverse effects for up to 1 year postadministration was observed. These findings reveal a wide safety margin for CERE-120 and collectively support the ongoing clinical testing of the efficacy and safety of CERE-120 in patients with Parkinsons disease.

Properly scaled and targeted AAV2-NRTN (neurturin) to the substantia nigra is safe, effective and causes no weight loss: support for nigral targeting in Parkinson's disease.

Recent analyses of autopsied brains from subjects previously administered AAV2-neurturin (NRTN) gene transfer argues that optimizing the effects of neurotrophic factors in Parkinsons disease (PD) likely requires delivery to both the degenerating cell bodies (in substantia nigra) and their terminals (in striatum). Prior to implementing this novel dosing paradigm in humans, we conducted eight nonclinical experiments with three general objectives: (1) evaluate the feasibility, safety and effectiveness of targeting the substantia nigra (SN) with AAV2-NRTN, (2) better understand and appraise recent warnings of serious weight loss that might occur with targeting the SN with neurotrophic factors, and (3) define an appropriate dose of AAV2-NRTN that should safely and effectively cover the SN in PD patients. Toward these ends, we first determined SN volume for rats, monkeys and humans, and employed these values to calculate comparable dose equivalents for each species by scaling each dose, based on relative SN volume. Using this information, we next injected AAV2-GFP to monkey SN to quantify AAV2-vector distribution and confirm reasonable SN coverage. We then selected and administered a ~200-fold range of AAV2-NRTN doses (and a single AAV2-GDNF dose) to rat SN, producing a wide range of protein expression. In contrast to recent warnings regarding nigra targeting, no dose produced any serious side effects or toxicity, though we replicated the modest reduction in weight gain reported by others with the highest AAV2-NRTN and the AAV2-GDNF dose. A dose-related increase in NRTN expression was seen, with the lower doses limiting NRTN to the peri-SN and the highest dose producing mistargeted NRTN well outside the SN. We then demonstrated that the reduction in weight gain following excessive-doses can be dissociated from NRTN in the targeted SN, and is linked to mistargeted NRTN in the diencephalon. We also showed that prior destruction of the dopaminergic SN neurons via 6-OHDA had no impact on the weight loss phenomenon, further dissociating neurotrophic exposure to the SN as the culprit for weight changes. Finally, low AAV2-NRTN doses provided significant neuroprotection against 6-OHDA toxicity, establishing a wide therapeutic index for nigral targeting. These data support targeting the SN with AAV2-NRTN in PD patients, demonstrating that properly targeted and scaled AAV2-NRTN provides safe and effective NRTN expression. They also provided the means to define an appropriate human-equivalent dose for proceeding into an ongoing clinical trial, using empirically-based scaling to account for marked differences in SN volume between species.

Enhanced neurotrophic distribution, cell signaling and neuroprotection following substantia nigral versus striatal delivery of AAV2-NRTN (CERE-120)

This paper reassesses the currently accepted viewpoint that targeting the terminal fields (i.e. striatum) of degenerating nigrostriatal dopamine neurons with neurotrophic factors in Parkinsons disease (PD) is sufficient for achieving an optimal neurotrophic response. Recent insight indicating that PD is an axonopathy characterized by axonal transport deficits prompted this effort. We tested whether a significantly greater neurotrophic response might be induced in SN neurons when the neurotrophic factor neurturin (NRTN) is also targeted to the substantia nigra (SN), compared to the more conventional, striatum-only target. While recognizing the importance of maintaining the integrity of nigrostriatal fibers and terminals (especially for achieving optimal function), we refocused attention to the fate of SN neurons. Under conditions of axonal degeneration and neuronal transport deficits, this component of the nigrostriatal system is most vulnerable to the lack of neurotrophic exposure following striatal-only delivery. Given the location of repair genes induced by neurotrophic factors, achieving adequate neurotrophic exposure to the SN neurons is essential for an optimal neurotrophic response, while the survival of these neurons is essential to the very survival of the fibers. Two separate studies were performed using the 6-OHDA model of nigrostriatal degeneration, in conjunction with delivery of the viral vector AAV2-NRTN (CERE-120) to continuously express NRTN to either striatum or nigra alone or combined striatal/nigral exposure, including conditions of ongoing axonopathy. These studies provide additional insight for reinterpreting past animal neurotrophic/6-OHDA studies conducted under conditions where axon transport deficiencies were generally not accounted for, which suggested that targeting the striatum was both necessary and sufficient. The current data demonstrate that delivering NRTN directly to the SN produces 1) expanded NRTN distribution within the terminal field and cell bodies of targeted nigrostriatal neurons, 2) enhanced intracellular neurotrophic factor signaling in the nigrostriatal neurons, and 3) produced greater numbers of surviving dopamine neurons against 6-OHDA-induced toxicity, particularly under the conditions of active axonopathy. Thus, these data provide empirical support that targeting the SN with neurotrophic factors (in addition to striatum) may help enhance the neurotrophic response in midbrain neurons, particularly under conditions of active neurodegeneration which occurs in PD patients.

Gene transfer provides a practical means for safe, long-term, targeted delivery of biologically active neurotrophic factor proteins for neurodegenerative diseases

Efforts to develop neurotrophic factors to restore function and protect dying neurons in chronic neurodegenerative diseases like Alzheimer’s (AD) and Parkinson’s (PD) have been attempted for decades. Despite abundant data establishing nonclinical proof-of-concept, significant delivery issues have precluded the successful translation of this concept to the clinic. The development of AAV2 viral vectors to deliver therapeutic genes has emerged as a safe and effective means to achieve sustained, long-term, targeted, bioactive protein expression. Thus, it potentially offers a practical means to solve those long-standing delivery/translational issues associated with neurotrophic factors. Data are presented for two AAV2 viral vector constructs expressing one of two different neurotrophic factors: nerve growth factor (NGF) and neurturin (NRTN). One (AAV2-NGF; aka CERE-110) is being developed as a treatment to improve the function and delay further degeneration of cholinergic neurons in the nucleus basalis of Meynert, the degeneration of which has been linked to cognitive deficits in AD. The other (AAV2-NRTN; aka CERE-120) is similarly being developed to treat the degenerating nigrostriatal dopamine neurons and major motor deficits in PD. The data presented here demonstrate: (1) 2-year, targeted, bioactive-protein in monkeys, (2) persistent, bioactive-protein throughout the life-span of the rat, and (3) accurately targeted bioactive-protein in aged rats, with (4) no safety issues or antibodies to the protein detected. They also provide empirical guidance to establish parameters for human dosing and collectively support the idea that gene transfer may overcome key delivery obstacles that have precluded successful translation of neurotrophic factors to the clinic. More specifically, they also enabled the AAV-NGF and AAV-NRTN programs to advance into ongoing multi-center, double-blind clinical trials in AD and PD patients.

947. Effects of Natural Pre-Existing Humoral Immunity to AAV on Administration of AAV2-NTN to the Brain of Non-Human Primates

The prevalence of antibodies (ab) to wild type AAV2 in humans is estimated to be 50-90%, with 30% developing neutralizing ab (Nab). In an attempt to mimic pre-existing immunity in animal models, many groups have immunized naive animals in order to study its effect on gene transfer. One limitation of this approach is that direct inoculation with high titers of AAV with adjuvant does not mimic the natural course of infection of the wild type virus. Therefore, the nature and intensity of the immune response may differ considerably from that generated by mucosal exposure. In addition, gene transfer is often attempted during the acute phase of the immune response. In contrast, human AAV2 seroconversion typically occurs in childhood, while AAV gene transfer is often performed in adults.