SOW Revised.pdf

Version 11-30-2016-1

FAR Part 11—Describing Agency Needs

STATEMENT OF WORK (SERVICES)

(SOW)

GENERAL INFORMATION

Title of Project:

Reprogramed iPSCs isolated from peripheral blood mononuclear cells of patients with MPD5.

Statement of Need and Purpose:

This requisition is to acquire Induced Pluripotent Stem Cell (iPSCs) Line Establishment Services for a for drug repurposing study for a rare disease called Distal 5 Myopathy (MPD5) caused by mutation in

ADSSL1 gene.

Background Information and Objective:

The NCATS Early Translation Branch (ETB) collaborates with investigators at NIH and in the academic, biopharmaceutical and nonprofit sectors to generate probes for studying a diverse cross-section of human biology, focusing specifically on new targets and untreatable diseases.

One of such untreatable diseases is Distal 5 Myopathy (MPD5) caused by mutation in ADSSL1 gene.

This is an ultra-rare disease which we could not be found on the National Organization for Rare Disorders

(NORD) or any other rare disease databases. It causes a rapid onset of progression on the spam of two years. It makes patients challenging to climb stairs along with increasing difficulty with activities like getting up from a chair. His walking, foot drop due to which they sometimes trip, they have difficulty swallowing, and experience shortness of breath while walking. They also have dysphagia with masticatory dysfunction, proximal muscle weakness, distal muscle weakness, heart muscle disease, reduced lung volume, muscle wasting (dystrophic changes), muscle cell death (necrotizing skeletal myopathy), rimmed vacuoles, Drooping eyelids (bilateral ptosis), dry eyes (keratoconjunctivitis sicca), facial muscle weakness (facial palsy), involuntary tight closure of the eyelids (blepharospasm), abnormal scarring (atrophic scars), stiff elbow joints (elbow flexion contracture), loss of ambulation

This genetic mutation adversely impacts the conversion of IMP to AMP in the Purine Nucleotide Cycle.

The goal is to conduct drug repurposing studies for MPD5 given that this myopathy results from impact on metabolic pathways. This requisition is to study further the biology of the mutation, which will help us develop compound screening campaigns.

Period of Performance:

6 months ARO.

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. Summary of Work. For the whole blood sample designated by ETB for reprogramming expansion, contractor will isolate peripheral blood mononuclear cells (PBMCs) and reprogram these cells into iPSCs as directed by the ETB. Initially, a Master Cell Bank (MCB) of one clone with at least three (3) vials will be created per PBMC submitted and stored.

Upon the successful creation of the MCB, two (2) vials will be shipped to ETB-specified collaborator location. One (1) additional vial of the MCB will be donated (submitted) to the National Institute for General Medical Sciences Human Genetic Cell Repository (NIGMS HGCR). These iPSCs will be subject to all regulations and terms of a submission to the

NIGMS HGCR.

2. Master Cell Bank – Generation and Quality Control.

2.1. MCB Deemed Successful. The MCB generation (expansion) will be deemed "successful" in respect of a iPSC line if (a) the number of vials of cells of such iPSC line specified above has been generated and (b) passed each MCB QC Test . In the event of a QC Test failure, another vial from the frozen MCB will be used to repeat the QC Test.

2.2. Master Cell Bank Quality Control Tests. As part of MCB creation activities contractor will perform the following QC Tests:

• SeV Genome RT-PCR: SeV Genome assay is a RT-PCR assay with SeV genome sequence and transgene-specific primers that allows specific detection of transgenes carried by CytoTune 2.0 Sendai Virus reprogramming vectors to confirm loss of expression of Sendai genome and transgenes.

• Viability: Viability is determined by measuring the area of five (5) colonies at first appearance (Day 1), then again within five (5) days. Colony doubling within five (5) days of the first appearance signifies a viable culture.

• Growth Rate: The MCB will be assessed for growth rate through colony doubling within five (5) days of first colony appearance.

• Mycoplasma: a sample of the MCB will be tested for mycoplasma using the MycoSEQ™ Mycoplasma Detection Kit from ThermoFisher Scientific. This real-time

PCR assay detects of >90 mycoplasma species with proven specificity and demonstrated sensitivity (detecting <10 copies per reaction).

• 6-Plex Short Tandem Repeat (STR): A sample of the MCB will undergo STR/identity analysis using Coriell’s 6-plex microsatellite assay. This is a short tandem repeat

(STR) multiplex PCR assay. This single reaction amplifies six (6) tetranucleotide repeat loci and amelogenin, the gender-determination marker.

• Karyotyping: Twenty (20) metaphases will be counted; of these at least five (5) cells will be analyzed and examined microscopically for numerical and structural abnormalities. At least five (5) cells will be karyotyped. Results include International

System for Human Cytogenetic Nomenclature (ISCN) results with at least two (2) images of G-banded karyograms. Harvested cell pellets will be kept until ETB accepts the MCB, slides with dropped metaphases for analysis will be kept for one month, and copies of the digital records will be available for three (3) years after the creation of such record. Contractor’s criteria for failure of karyotype testing shall be defined by any of the following results:

o Of the initial twenty (20) cells that are counted and five (5) cells that are analyzed, if three (3) of the analyzed cells show the same abnormality, then ten (10) additional cells will be assessed. If one (1) more cell, for a total of 4 analyzed cells, contains the same abnormality, then the line is deemed abnormal.

o If contractor deems an iPSC line to have failed Karyotyping, ETB shall have the option to request an additional karyotyping QC test at an additional cost. The parties agree to discuss in good faith any karyotyping QC issues and any potential resolution prior to an iPSC line to be deemed to have failed Karyotyping.

o If a certain abnormality was found in a previous study (parent line, sister line, or expansion), the line fails.

o If the total non-clonal abnormalities ≥25%, the line fails.

• SNP 6.0 Genotyping: a MCB sample will be analyzed using the Affymetrix Genome- Wide Human SNP Array 6.0 (SNP CGH Array) in CORIELL's genotyping center. Upon detection of cytogenomic abnormalities, lines will fail at the discretion of the cytogenomics consultant.

• Embryoid Body (EB) Formation from Undifferentiated Cells and RT-PCR to confirm trilineage differentiation potential: EB formation assay will be conducted using undifferentiated iPS cells and it will be followed for ten (10) days in culture. After ten

(10) days, embryoid bodies (EBs) will be harvested followed by RNA extraction, cDNA synthesis, and RT-PCR assay to detect the expression of genes in all three (3) germ layers. Expression of at least one gene per germ layer is required for confirming pluripotency.

• Alkaline Phosphatase (AP): Staining will be conducted using the StemTAG Alkaline Phosphatase Staining kit from Cell Biolabs to confirm that the culture is within the desired limit of undifferentiated stem cells as a high level of AP and high AP activity are traditional markers of pluripotent stem cells.

• Cell Surface Antigen Expression: Analysis will be conducted by flow cytometry using SSEA 3 and SSEA 4 staining. Flow cytometry is also performed if an abnormal morphology is observed throughout the culturing process in order to confirm that the culture is within the desired limit of undifferentiated iPS cells.

3. Storage, Distribution, and Destruction of MCB. Two (2) vials of successfully established MCB vials will be shipped to ETB or ETB-desired recipient upon completion.

LEVEL OF EFFORT:

Scientists with specialized training in iPSCs shall conduct the experiment alongside the main investigator.

GOVERNMENT RESPONSIBILITIES

The NCATS Early Translation Branch (ETB) and its collaborators will provide vendor with whole blood sample designated for reprogramming expansion.

DELIVERY OR DELIVERABLES

Contractor shall deliver within 6 months ARO:

1. Two (2) vials of reprogramed iPSCs isolated from peripheral blood mononuclear cells (PBMCs)

2. One (1) additional vial of the MCB will be donated (submitted) to the National Institute for

General Medical Sciences Human Genetic Cell Repository (NIGMS HGCR). These iPSCs will be subject to all regulations and terms of a submission to the NIGMS HGCR.

REPORTING REQUIREMENTS

iPSCs quality control assessments/tests

OTHER CONSIDERATIONS

Travel:

N/A

Key Personnel:

Information System Security Plan:

Data Rights:

Section 508—Electronic and Information Technology Standards:

Publications and Publicity:

Confidentiality of Information: