B. SOW Updated.pdf

Version 11-30-2016-1 FAR Part 11—Describing Agency Needs

STATEMENT OF WORK (SERVICES)

(SOW)

GENERAL INFORMATION

Title of Project:

NCATS Project Phase II Supplement – Target Validation and therapeutic evaluation in NGLY1 deficient chip-based models.

Statement of Need and Purpose:

The purpose of this requirement is to obtain an evaluation of therapeutic targets for drug compound evaluation by siRNA knockdown of ENGase or lentiviral transfection of NGLY1 to ameliorate the NGLY1 mediated phenotypes in motoneurons and neuromuscular junction (NMJ) models.

Background Information and Objective:

The National Center for Advancing Translational Sciences (NCATS) focuses on getting more treatments to more patients more quickly. Several thousand genetic diseases affect humans, of which only about 500 have any treatment. NCATS is directly addressing this problem by discovering new technologies and other approaches that could greatly accelerate the process of developing and deploying solutions that can be used by all translational researchers.

NGLY1 deficiency include abnormal tear production, a movement disorder, and liver disease.

Additionally, it includes developmental delay, hypotonia, peripheral neuropathy, EEG abnormalities, and microcephaly.

The Therapeutic Develop Branch (TDB) program’s mission is to encourage and speed the development of new treatments for diseases with high-unmet medical needs. NGLY1 deficiency is greatly involved with motor neurons malfunction and therefore NCATS has been collaborating with Hesperos Inc., in phase 1 to differentiate all motor neurons from NGLY1 patient lines.

Phase 1 of the project resulted in interesting patch clamp and NMJ data indicating differences in

NGLY1 mutated motoneurons, a KO phenotype and wild type motoneurons. Neuromuscular junction analysis data indicated a range of deficiencies depending on the motoneuron mutation including decreases in fidelity and stability. Currently NCATS is in phase 2 of the project and evaluation of therapeutic targets for drug compound evaluation by siRNA knockdown of

ENGase or lentiviral transfection of NGLY1 to ameliorate the NGLY1 mediated phenotypes in motoneurons and neuromuscular junction (NMJ) models. NCATS requires an evaluation of therapeutic targets for drug compound TRND00601748-01 for three patient lines of NGLY1 to ameliorate the NGLY1 mediated phenotypes in motoneurons in patch clamp and neuromuscular junction (NMJ) models.

Period of Performance:

The period of performance is four (4) months from time of award.

Place of Performance:

Services to be performed at Contractor on-site facility

SCOPE OF WORK

General Requirements:

Independently and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government as needed to perform the Statement of Work below:

Specific Requirements:

1. The Contractor shall perform a theory for experimental design, specifically, The deficiencies in patch clamp electrophysiology are relatively uniform across all three diseased lines. Each line possesses a different LOF mutation in NGLY1, each of which could contribute differently to the diseased phenotype noted in phase 1 of the project.

Therefore, NCATS will use all three previously differentiated diseased lines to validate the effects of modulating ENGase and NGLY1 expression in mature motoneurons through patch clamp electrophysiology readouts and neuromuscular physiology.

NCATS will provide the iPSC Motoneurons for the theory for experimental design and the NMJ system analysis:

1. Wild Type (WT) Control (268A)

2. Isogenic NGLY1 knockout (X2-9)

3. NGLY1 patient 1 sample (592D)

4. NGLY1 patient 2 sample (594A)

Milestone 0a: Phase 2: The Contractor shall test the NCATS hit compound

TRND00601748-01 and additionally in all three diseased lines in addition to WT line after change from fast acting to slow acting drugs

For testing performed for Experiment 1, the Contractor shall measure the changes in patch clamp electrophysiology readouts of TRND00601748-01 at one timepoint post dosing (24-hours) at 5 concentrations in one diseased line.

Cultures will be tested in singlet per experiment across 3 biological repeats.

Motoneurons derived from diseased lines (X2-9) will be dosed at 5 different concentrations of a drug and measured at 24-hours post dosing. A dose response curve for each line will be built for the following patch-clamp parameters:

• Ion channel currents (Including Na+ and K+)

• Spontaneous action potential firing frequency

• Resting membrane potential

Results shall be delivered by the Contractor after these experiments.

For testing performed for Experiment 2, the Contractor shall measure the changes in NMJ function of TRND00601748-01 at one timepoint post dosing (24-hours) at 5 concentrations in all three diseased lines. Cultures will be tested in singlet per experiment across 3 biological repeats. Motoneurons derived from diseased lines (X2-9, 594A, and 592D) will be cultured with healthy skeletal muscle in Hesperos’ NMJ chambers. These cultures will be dosed at 5 different concentrations of a drug and measured at 24-hours post dosing. A dose response curve for each line will be built for the following NMJ parameters:

2. The Contractor shall perform an NMJ system analysis, specifically,

1. Total number of NMJs in each system.

2. Stability of NMJs: NMJ stability is defined as the ratio of total number of NMJs per system post-stimulation to the number of NMJs pre-stimulation.

3. Synchronous firing of a specific NMJ: One NMJ tested at three different frequencies

(0.33, 0.5, and 1Hz). The percentage of successful muscle contractions driven by electrically-induced MN action potential generation will be quantified.

Milestone 0b: Phase 2:

I. Due to changes in the experimental plan of Phase II from two slow-acting and two fast acting compounds to 4 slow-acting compounds the fast-acting compounds will be replaced with one slow acting compound (CREB-activator: 8-Bromo-cAMP) in all three diseased lines and one slow acting compound (NKCC1 inhibitor: Bumetanide) in WT cells only.

Milestone 2: Optimize the siRNA transfection and lentiviral transfection in diseased lines.

The concentration or titer as well as ideal exposure time will be optimized for each vector and will be determined by analysis of ENGase and NGLY1 expression levels. NCATS will provide Hesperos with the necessary PCR primer sequences as well as any relevant information they may have regarding starting dose concentrations and incubation times.

Milestone 2 will commence upon receipt of viral vectors and PCR primer sequences.

LEVEL OF EFFORT:

Project Timeline:

- Milestone 0a: two months

- Milestone 0b and 2: two months

GOVERNMENT RESPONSIBILITIES

Government will be responsible for providing any potential iPSC lines needed and potential compounds to be tested to the Contractor.

DELIVERY OR DELIVERABLES

Milestone 2 will commence upon receipt of viral vectors and PCR primer sequences. The transition to and start of future milestones will be at the sole discretion of NCATS and will only commence upon agreement from all parties.

REPORTING REQUIREMENTS

The Contractor shall deliver to TDB a complete data package including spreadsheet of raw data (viability counts, immunocytochemistry quantification, etc.) as well as a complete data work-up and full report for four patient lines and the compounds being tested.

OTHER CONSIDERATIONS

Travel:

Not Applicable

Key Personnel:

Not Applicable

Information System Security Plan:

Not applicable

Data Rights:

All data produced, used and analyzed is sole property of the government. Contractor has no right to do any analysis with iPSCs lines provided by TDB rather than indicated in SOW and must keep all the data and information confidential. Contractor has no right to use the assay and system developed for our project with our cell lines and differentiated lines for any other project.

Section 508—Electronic and Information Technology Standards:

Not applicable

Publications and Publicity:

NCATS will collaborate with Hesperoc Inc. to publish another paper for Target Validation and therapeutic evaluation in NGLY1 deficient chip-based models.

Confidentiality of Information:

All the data will be confidential.