Anne M. Douar

Intravenous Administration of Self-complementary AAV9 Enables Transgene Delivery to Adult Motor Neurons

Therapeutic gene delivery to the whole spinal cord is a major challenge for the treatment of motor neuron (MN) diseases. Systemic administration of viral gene vectors would provide an optimal means for the long-term delivery of therapeutic molecules from blood to the spinal cord but this approach is hindered by the presence of the blood-brain barrier (BBB). Here, we describe the first successful study of MN transduction in adult animals following intravenous (i.v.) delivery of self-complementary (sc) AAV9 vectors (up to 28% in mice). Intravenous MN transduction was achieved in adults without pharmacological disruption of the BBB and transgene expression lasted at least 5 months. Importantly, this finding was successfully translated to large animals, with the demonstration of an efficient systemic scAAV9 gene delivery to the neonate and adult cat spinal cord. This new and noninvasive procedure raises the hope of whole spinal cord correction of MN diseases and may lead to the development of new gene therapy protocols in patients.

Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10¹¹ particles/ml; 95% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹⁰ vector genomes/ml; 95% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95% CI, 2.06 x 10⁹ to 9.26 x 10⁹ TCID₅₀ IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.

Sustained delivery of therapeutic concentrations of human clotting factor IX--a comparison of adenoviral and AAV vectors administered in utero.

Prenatal somatic gene therapy has been considered for genetic disorders presenting with morbidity at birth. Haemophilia is associated with an increased risk of catastrophic perinatal bleeding complications such as intracranial haemorrhage, which could be prevented by gene transfer in utero. Prenatal gene therapy may be more promising than postnatal treatment, as the fetus may be more amenable to uptake and integration of therapeutic DNA and the immaturity of its immune system may permit life‐long immune tolerance of the transgenic protein, thus avoiding the dominant problem in haemophilia treatment, the formation of inhibitory antibodies.

Reversal of Type 1 Diabetes by Engineering a Glucose Sensor in Skeletal Muscle

Type 1 diabetic patients develop severe secondary complications because insulin treatment does not guarantee normoglycemia. Thus, efficient regulation of glucose homeostasis is a major challenge in diabetes therapy. Skeletal muscle is the most important tissue for glucose disposal after a meal. However, the lack of insulin during diabetes impairs glucose uptake. To increase glucose removal from blood, skeletal muscle of transgenic mice was engineered both to produce basal levels of insulin and to express the liver enzyme glucokinase. After streptozotozin (STZ) administration of double-transgenic mice, a synergic action in skeletal muscle between the insulin produced and the increased glucose phosphorylation by glucokinase was established, preventing hyperglycemia and metabolic alterations. These findings suggested that insulin and glucokinase might be expressed in skeletal muscle, using adeno-associated viral 1 (AAV1) vectors as a new gene therapy approach for diabetes. AAV1-Ins+GK–treated diabetic mice restored and maintained normoglycemia in fed and fasted conditions for >4 months after STZ administration. Furthermore, these mice showed normalization of metabolic parameters, glucose tolerance, and food and fluid intake. Therefore, the joint action of basal insulin production and glucokinase activity may generate a “glucose sensor” in skeletal muscle that allows proper regulation of glycemia in diabetic animals and thus prevents secondary complications.

Therapeutic plasma concentrations of human factor IX in mice after gene delivery into the amniotic cavity: a model for the prenatal treatment of haemophilia B

Several groups including our own have reported gene delivery to fetal organs by vector administration into the amniotic cavity. Based on these studies we hypothesised that the large surface of the fetal skin may be exploitable for high level production of systemically required gene products to be released into the fetal circulation.

Deleterious effect of peptide insertions in a permissive site of the AAV2 capsid

A permissive site for insertion of heterologous peptide sequences has been identified in the capsid proteins of AAV2. While attempting to use this site for insertion of a nuclear localization sequence, we have observed a drastic reduction in the yield of DNA-containing particles. ELISA analysis showed that capsid assembly was modestly affected, whereas genome encapsidation was more profoundly altered, a phenomenon we did not observed when a RGD peptide was inserted at the same location. Furthermore, the NLS viruses displayed poor transduction efficiency on HeLa and 293 cells. Altogether, these results indicate that the nature of the peptide inserted at position 587 in the capsid may have important consequences on both particle formation and infectivity.

268. Optogenetic Engineering of Retinal Ganglion Cells with AAV2.7m8-ChrimsonR-tdTomato (GS030-DP) Is Well Tolerated and Induces Functional Responses to Light in Non-Human Primates

Introduction: Expression of a light-sensitive opsin in retinal ganglion cells (RGCs) is an attractive strategy to restore vision. We evaluated the ability of ChrimsonR-tdTomato (ChrR-tdT), derived from the algal light-gated cation channel ChrimsonR (Ed Boyden, MIT), to convert light insensitive RGCs into photoactivatable cells in normal macaques. A photostimulation device (GS030-MD) is developed in parallel to complement the biologics. This GS030 combination treatment is intended to treat blindness caused by retinal degenerative diseases such as retinitis pigmentosa. Methods: Cynomolgus macaques were injected intravitreally with the AAV2.7m8 vector encoding ChrR-tdT under the control of the CAG promoter (GS030-DP; 5×1011 vg/eye). Electrophysiological measurements by microelectrode array (MEA) and patch clamp as well as expression of the ChrR-tdT protein by immunofluorescence were assessed on explanted retinas 2 months after injection. Local tolerance was evaluated by ophthalmic examination and histology at 2 and 6 months post administration. Results: ChR-tdT was essentially expressed in RGCs and its expression restricted to the perifoveal area. MEA recordings showed light responses in all treated retinas, with 3 out of 4 retinas displaying high amplitudes of electrical responses to light stimulation (up to 360 Hz). One retina was less responsive (50 Hz). In patch clamp experiments, conducted by targeting tdT-expressing RGCs, large photocurrents were recorded in 3 out of 4 retinas in response to illumination, and according to the expected action spectrum for ChR-tdT. An exploratory study was conducted in parallel in monkeys, which received a single bilateral intravitreal administration of GS030-DP (5×1011 vg/eye) to assess local tolerance, systemic toxicity and immunogenicity at 2 and 6 months. No clinical signs indicative of systemic toxicity or local intolerance were observed. No adverse effects were seen by ophthalmic or histological examinations, especially no retinal structural modifications, inflammation or necrosis. Anti-AAV2 neutralizing antibodies (NAbs) measured in serum at 2, 3 and 6 months slightly increased at month 2 (≤ 1:100) and then returned to baseline levels at month 6. No NAbs were detected in aqueous humor at necropsy (at 2 or 6 months). In parallel, in preparation of a first-in-man clinical trial, a complete prototype of the photostimulation device (“goggles”) was developed with a full functional optical chain, an electronic subsystem, and firmware and software architecture. These goggles (GS030-MD) capture external scenes through an event-based camera and deliver visual stimuli onto the transduced retina at irradiances shown to activate ChrR-tdT expressing RGCs in monkey retinas. Conclusion: GS030 vector (GS030-DP) transduced efficiently and safely RGCs in vivo after intravitreal administration and induced light responses in normal monkey retinas under pharmacological block of endogenous phototransducion. The GS030 treatment combining the AAV2.7m8-ChrR-tdT vector and the photo stimulation goggles represents a valuable treatment for vision restoration in retinitis pigmentosa.

794. Evaluation of Gene Transfer Efficacy Mediated by AAV1 and AAV6 Vectors in Skeletal Muscle of Adult Mice Following Different Routes of Administration

Top of pageAbstract Skeletal muscle represents an important target tissue for gene therapy application due to the large number of genetic muscular disorders including Duchene Muscular Dystrophy. In addition the muscle may be also used as a delivery platform to express therapeutic proteins into the bloodstream for non-muscular metabolic disorders. Adeno-Associated Virus 1-based vector (rAAV1) is often considered as the most efficient AAV serotype to deliver genes into the muscles. In this study, we compared the performance of AAV pseudotyped (based on AAV2 genome) vectors expressing a secreted form of the murine alkaline phosphatase, (mSEAP) with capsid from serotype 1 and 6, in mouse skeletal muscle using different delivery methods: direct injection into individual muscle (intramuscular, IM), local limb distribution of vectors via the circulation (intra-arterial delivery, IA) or systemic delivery (intravenous administration, IV). We injected the maximal volume that each of these route allowed. We analyzed two parameters: the levels of a reporter circulating protein (), a relevant parameter for a depot organ strategy, and the extent of fiber transduction in the muscles, a relevant parameter in view of a muscular disorder therapeutic approach. We compared rAAV1 and rAAV6 encoding mSEAP in a dose response study following IM administration in adult C57Bl/6 mice. Both vectors showed strong and equivalent levels of transduction at the highest doses (6x108 vg and 3x109 vg), while at the lowest dose injected (1.5x1010 vg), rAAV6 was 3-fold more efficient than rAAV1 in transducing muscle, suggesting a differential threshold of efficiency at sub-optimal doses. In addition, the potential for systemic gene transfer after IV of rAAV1 and rAAV6 at the whole body level was investigated. For the two doses tested (1|[times]|1011 vg and 3|[times]|1011 vg). AAV6 vector led to 3-fold higher levels of circulating mSEAP levels than rAAV1. In addition, a widespread transduction of both skeletal and cardiac tissues was observed with rAAV6 after histochemical detection of mSEAP. However, in term of circulating protein level, IV administration was less efficient than IM injection since the same level of circulating mSEAP was achieved with 3|[times]|1011 vg using IV delivery compared to 3|[times]|109 vg for IM delivery. We have also evaluated intra-arterial delivery via the femoral artery of rAAV1 and rAAV6 at two doses (1|[times]|1011 vg and 3|[times]|1011 vg). This route yielded a 10 fold higher mSEAP levels in the serum than IV and led to robust transgene expression pattern in the hind limb muscles. Finally, in an attempt to increase gene transfer efficacy in muscle after IV delivery, we co-injected the VEGF, with either rAAV1 or rAAV6. For both vectors, the presence of VEGF did not show a positive impact on transgene expression levels. In conclusion, our results show that despite the high similarity between AAV1 and AAV6 capsid sequences, rAAV6 performance for muscle transduction are superior to rAAV1 with all IM, IA or IV delivery routes. This finding prompts us to consider AAV6 vectors as one of the most efficient viral agent to deliver gene in skeletal muscle and further clinical applications.