Brian D. Cleaver

Pompe disease gene therapy

Pompe disease is an autosomal recessive metabolic myopathy caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase and results in cellular lysosomal and cytoplasmic glycogen accumulation. A wide spectrum of disease exists from hypotonia and severe cardiac hypertrophy in the first few months of life due to severe mutations to a milder form with the onset of symptoms in adulthood. In either condition, the involvement of several systems leads to progressive weakness and disability. In early-onset severe cases, the natural history is characteristically cardiorespiratory failure and death in the first year of life. Since the advent of enzyme replacement therapy (ERT), the clinical outcomes have improved. However, it has become apparent that a new natural history is being defined in which some patients have substantial improvement following ERT, while others develop chronic disability reminiscent of the late-onset disease. In order to improve on the current clinical outcomes in Pompe patients with diminished clinical response to ERT, we sought to address the cause and potential for the treatment of disease manifestations which are not amenable to ERT. In this review, we will focus on the preclinical studies that are relevant to the development of a gene therapy strategy for Pompe disease, and have led to the first clinical trial of recombinant adeno-associated virus-mediated gene-based therapy for Pompe disease. We will cover the preliminary laboratory studies and rationale for a clinical trial, which is based on the treatment of the high rate of respiratory failure in the early-onset patients receiving ERT.

Preclinical potency and safety studies of an AAV2-mediated gene therapy vector for the treatment of MERTK associated retinitis pigmentosa.

Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

A simplified purification protocol for recombinant adeno-associated virus vectors

We describe a new rapid, low cost, and scalable method for purification of various recombinant adeno-associated viruses (rAAVs) from the lysates of producer cells of either mammalian or insect origin. The method takes advantage of two general biochemical properties of all characterized AAV serotypes: (i) low isoelectric point of a capsid and (ii) relative biological stability of the viral particle in the acidic environment. A simple and rapid clarification of cell lysate toremove the bulk of proteins and DNA is accomplished by utilizing inexpensive off-the-shelf reagents such as sodium citrate and citric acid. After the low-speed centrifugation step, the supernatant is subjected to cation exchange chromatography via sulfopropyl (SP) column. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice (GMP) grade biochemical purity. The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format.

Evaluation of Readministration of a Recombinant Adeno-Associated Virus Vector Expressing Acid Alpha-Glucosidase in Pompe Disease: Preclinical to Clinical Planning

A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon-optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e., rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early- and late-onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be readministered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies that may reduce the effectiveness of the vector, especially if readministration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa(-/-) mice, an animal model of Pompe disease. This article describes the IND-enabling preclinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for readministration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with late-onset Pompe disease will be enrolled. The goal of the immune modulation strategy is to ablate B-cells before the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of the active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the evidence generated from gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the three months following each dosing will assess the safety, biochemical, and functional impact of the vector.

The effect of pulsatile gonadotropin-releasing hormone and estradiol administration on luteinizing hormone and follicle-stimulating hormone concentrations in pituitary stalk-sectioned ovariectomized pony mares☆

Hourly pulses of gonadotropin-releasing hormone (GnRH) or bi-daily injections of estradiol (E2) can increase luteinizing hormone (LH) secretion in ovariectomized, anestrous pony mares. However, the site (pituitary versus hypothalamus) of positive feedback of estradiol on gonadotropin secretion has not been described in mares. Thus, one of our objectives involved investigating the feedback of estradiol on the pituitary. The second objective consisted of determining if hourly pulses of GnRH could re-establish physiological LH and FSH concentrations after pituitary stalk-section (PSS), and the third objective was to describe the declining time trends of LH and FSH secretion after PSS. During summer months, ovariectomized pony mares were divided into three groups: Group 1 (control, n = 2), Group 2 (pulsatile GnRH (25 micrograms/hr), n = 3), and Group 3 (estradiol (5 mg/12 hr), n = 3). All mares were stalk-sectioned and treatment begun immediately after stalk-section. Blood samples were collected every 30 min for 8 h on the day before surgery (D0) and 5 d post surgery (D5) to facilitate the comparison of gonadotropin levels before and after pituitary stalk-section. Additionally, jugular blood samples were collected every 12 hr beginning the evening of surgery, allowing for evaluation of the gonadotropin secretory time trends over the 10 d of treatment. On Day 10, animals were euthanized to confirm pituitary stalk-section and to submit tissue for messenger RNA analysis (parallel study). Plasma samples were assayed for LH and FSH by RIA. Mean LH secretion decreased from Day 0 to Day 5 in Groups 1 and 3, whereas LH secretion tended (P < 0.08) to decrease in Group 2 mares. On Day 5, LH was higher (P < 0.01) in Group 2 (17.26 +/- 3.68 ng/ml: LSMEANS = SEM), than either Group 1 (2.65 +/- 4.64 ng/ml) or Group 3 (4.28 +/- 3.68 ng/ml). Group 1 did not differ from Group 3 on Day 5 (P < 0.40). Similarly, mean FSH levels decreased in all groups after surgery, yet Group 2 mares had significantly (P < 0.001) higher FSH concentrations (17.66 +/- 1.53 ng/ml) than Group 1 or Group 3 (8.34 +/- 1.84 and 7.69 +/- 1.63 ng/ml, respectively). Regression analysis of bi-daily LH and FSH levels indicated that the time trends were not parallel. These findings indicate: 1) Pituitary stalk-section lowered LH and FSH to undetectable levels within 5 d after surgery. 2) pulsatile administration of GnRH (25 micrograms/hr) maintained LH and FSH secretion, although concentrations tended to be lower than on Day 0, and 3) E2 did not stimulate LH or FSH secretion.

A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform.

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.

EFFECTS OF OVARIAN INPUT ON GnRH AND LH SECRETION IMMEDIATELY POSTOVULATION IN PONY MARES

The potential involvement of ovarian factors in regulating GnRH and LH postovulation was studied in ovarian intact (Group 1; n=3) and ovariectomized (OVX; Group 2; n=3) mares (OVX within 12 hr of ovulation). Blood samples were collected every 10 min for 6 hr from jugular vein (JV) and intercavernous sinus (ICS) during estrus and on Day 8 postovulation for LH and GnRH analysis. Additionally, JV samples were collected twice daily (12-hr intervals) for 30 days for LH and progesterone (P4) analysis. A significant treatment x day effect (P<0.0001) describes declining plasma LH concentrations in intact mares, and regression analysis indicated that response curves were not parallel (P<0.001). Plasma LH concentrations remained elevated in OVX mares. LH increased further in OVX mares by Day 8 post-OVX (P<0.06), reflecting the increased (P<0.07) LH episode amplitude. GnRH decreased from estrus to Day 8 in both groups reflecting an effect of sampling period (P<0.03). GnRH episode amplitude declined (P<0.08) from estrus (62.8+/-3.1 pg/mL) to Day 8 (46.3+/-3.1 pg/mL) in OVX mares, but not in control mares (intact estrus, 36.5+/-6.4; intact Day 8, 37.5+/-7.3; OVX estrus, 62.8+/-3.1; OVX Day 8, 46.3+/-3.1 pg/mL). In conclusion, we propose that postovulatory LH decline requires ovarian feedback in mares, and that OVX alters GnRH secretory dynamics such that LH concentrations does not decline postovulation and, in fact, is further elevated with time after OVX.

472. Evaluation of Re-Administration of a Recombinant Adeno-Associated Vector Expressing Acid-Alpha-Glucosidase (rAAV9-DES-hGAA) in Pompe Disease: Preclinical to Clinical Planning

A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e. rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early and late onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be re-administered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies (NAb) that may reduce the effectiveness of the vector, especially if re-administration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa−/− mice, an animal model of Pompe disease. We describe the IND-enabling pre-clinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for re-administration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior (TA) muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with Late-Onset Pompe Disease (LOPD) will be enrolled. The goal of the immune modulation strategy is to ablate B-cells prior to the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the approach to gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the 3 months following each injection, will assess the safety, biochemical, and functional impact of the vector.View Large Image | Download PowerPoint Slide