Evaluation of adeno-associated virus gene therapy in the feline model ofmucolipidosis II

Progress Report December 2017
Statement of Significance: The combination of efficacy and safety data on
intravenous AAV9 gene therapy from animal models and the preliminary datafrom human trials are highly encouraging. We therefore believe that intravenous
delivery of AAV9 holds the strongest potential for effectively treating ML II andshould be evaluated in the feline model of ML II.
Specific Aim 1: Generation and preliminary characterization of AAV vector
encoding feline GNPTAB.
The design and production of the AAV vector encoding the feline GNPTAB gene
will be under the direction of Dr. Steven Gray (co-investigator) at the University ofNorth Carolina School of Medicine Gene Therapy Center. Dr. Gray has extensive
experience producing and optimizing therapeutic AAV vectors for a wide variety
of diseases, including numerous lysosomal storage disorders. Dr. Gray has also
successfully completed pre-clinical IND-enabling studies that resulted in a clinical
trial using AAV9 as a therapy for giant axonal neuropathy (NCT02362438).
1.1 A codon optimized version of the 3.8 kb feline GNPTAB gene will besynthesized and cloned into an AAV vector expression cassette, utilizing theCBh promoter which has been shown to confer strong non-specificexpression (13, 14). This will be transfected into HeLa cells to verify
expression of GNPTAB.
1.2 The GNPTAB construct will then be packaged in AAV9 and injected
intravenously (20 uL, 1x1012 vg total) into 6 neonatal wild type mice (3 male, 3female) to simulate a “worst-case” overexpression scenario in tissues such as
the liver that will receive a high vector load. Six control littermates will receive
vehicle injections. The mice will be weighed and observed weekly for overt
signs of adverse effects. At 2, 4, and 6 months old blood will be collected toassess possible chronic changes in serum chemistry (ALT, AST, BUN,albumin, CK) in response to gene transfer. If any adverse effects areapparent, mice will be sacrificed at 6 months (or when they reach a humane
euthanasia endpoint) to allow histopathology of all major organs. If the mice
maintain overall good health this will be done at 12 months old.

Progress in the current reporting period:
As proposed in Aim 1.1 a codon optimized version of the 3.8 kb feline GNPTAB
gene was synthesized and cloned into an AAV vector expression cassette
utilizing the CBh promoter, which is a promoter that confers strong non-specificexpression of the transgene it is driving. The newly created Cbh-GNPTABoptsPa construct was then transfected in to MLII fibroblast cells to confirm GNPTAB
protein expression. Unexpectedly, we saw significant cell death in the viraltransfected MLII fibroblast cells. The same Cbh-GNPTABopt-sPa construct wasthen transfected in to MLII HeLa cells to analyze GNPTAB expression andtoxicity in a second cell line. Interestingly, this construct was not toxic to MLII
HeLa cells suggesting that the toxicity seen in the fibroblasts could be cell line
specific. However, due to the apparent toxicity, a second vector construct wasmade in which a JeT+I promoter, a weaker promoter than the previous Cbh,
drove the GNPTAB gene. The second construct design was Jet+I-GNPTABoptSV40pA.

As proposed in Aim 1.2, with the GNPTAB constructs completed we proceeded
to packaging the constructs in AAV9 capsids. Small 0.5L lots of vector weremade for both the Cbh-GNPTABopt-sPa and Jet+I-GNPTABopt-SV40pA
constructs in to an AAV9 capsid. Vector production for both constructs wasrecently completed and 2 cohorts of wild type mice have now be treatedintravenously with ~4E11 vector genomes of 1 of the constructs, AAV9-CbhGNPTABopt-sPa or AAV9-Jet+I-GNPTABopt-SV40pA. The mice are currently
being regularly weighed and observed for any signs of overt toxicity. Blood will
also be assessed for any possible chronic changes in serum chemistry (ALT,AST, BUN, albumin, CK) in response to gene transfer. Lastly, the mice will besacrificed (the first group in mid-December) and assessed for liver toxicity. Basedon the short-term (2-week) toxicity results, we will decide if one or bothconstructs are safe to inject more mice for longer-term toxicity evaluation. If wesee liver toxicity with both of our current constructs, we will repackage theconstructs in to a liver-detargeting vector as described in the anticipated results,barriers, and alternative section of the grant. Based on the safety and toxicity
data from the mouse study, we will proceed with making a larger batch of thebest vector for treatment of MLII cats.

Plans for next reporting period:
We anticipate having clinical and postmortem data from mice treated with bothconstructs by the end of January 2018. At this time we will proceed withadditional long-term toxicity studies in mice and begin vector production for theMLII cats. The feline MLII breeding colony is still being maintained at theUniversity of Pennsylvania School of Veterinary Medicine. The apparent toxicityof the first construct with the intended Cbh promoter necessitated creation of asecond vector construct. Since two constructs had to be made, packaged in toAAV, and tested in mice, this has resulted in some delays. However, we still
anticipate being able to treat all proposed 6 MLII cats (Aim 2) by the end of thenext funding period.

Lay summary

The combination of efficacy and safety data on intravenous AAV9 gene therapy
from animal models and the preliminary data from human trials are highly
encouraging. We therefore believe that intravenous delivery of AAV9 holds thestrongest potential for effectively treating ML II and should be evaluated in thefeline model of ML II. Aim 1 of this grant proposal entailed generation andpreliminary characterization of AAV vector encoding feline GNPTAB. During thisreporting period we have created two AAV9 vector constructs, AAV9-CbhGNPTABopt-sPa and AAV9-Jet+I-GNPTABopt-SV40pA, the first driving astronger level of expression of the GNPTAB protein and the second a weakerlevel of expression. Two cohorts of wild type mice have now be treatedintravenously with 1 of the constructs, AAV9-Cbh-GNPTABopt-sPa or AAV9Jet+I-GNPTABopt-SV40pA, and are currently being evaluated for safety andtoxicity. Once safety and toxicity studies in mice are completed, large-scale
vector manufacturing will begin to treat MLII cats in the next reporting period.