D_SOW.pdf

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

STATEMENT OF WORK

(SOW)

GENERAL INFORMATION

Title of Project:

Custom Cloning and lentiviral preparation services for the development of assays and therapeutics for NGYL1 disease for the Therapeutic Development Branch (TDB) at the National Center for Advancing Translational Sciences (NCATS).

Statement of Need and Purpose:

This request is for a service contract for custom cloning and lentiviral preparation services, to be used in the TDB-Biology laboratory at NCATS. The purpose of the service is to obtain high quality lentiviral particles containing specific genes of interest for the NGLY1 disease. These lentiviral preparations will be used in cell-based assay development and for testing feasibility of gene therapy-based approaches for NGLY1 disease.

Background Information and Objective:

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.

Within NCATS, the TDB biology group works to encourage and speed the development of new treatments for diseases with high-unmet medical needs. This acquisition specifically addresses the new therapeutic for NGLY1 rare disease project, which specifically applies breakthroughs in translational science to advancing new treatments for rare diseases.

TDB biology is requesting a service to clone genes of interest into lentiviral vector and lentiviral preparations for the same. The contractor will custom clone three different types of constructs, prepare mega-scale endo-free DNA preparation as well as lentiviral particles at >10^9 IFUs/mL.

These lentiviral preparations will be used at NCATS to test two different therapeutic approaches for the NGLY1 disease:

1. One of the constructs and its lentiviral preparation will be used to develop a cell-based assay adapted from researchers at Yale University.

2. Two other constructs and their lentiviral preparation will be used to test potential RNA therapy for the NGLY1 disease.

Period of Performance:

The period of performance of this contract will be 3 months from the date of contract award.

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:

Summary

1. PCR cloning of the NGLY1 from pcDNA3.1-Hygro-NGLY1 template into pCDH-CMV-MCS- EF1α-GreenPuro (Ampicillin) via 5’ NheI and 3’ NotI by recombination, with sequence verification across the insert (1985bp); making NGLY1 in pCDH-CMV-MCS-EF1α-GreenPuro and delivered as an endo-free mega-scale DNA sample.

2. PCR cloning of the NGLY1-Del8 from pcDNA3.1-Hygro-NGLY1-Del8 template into pCDH- CMV-MCSEF1α-GreenPuro (Ampicillin) via 5’ NheI and 3’ NotI by recombination, with sequence verification across the insert (1874bp); making NGLY1-Del8 in pCDH-CMV-MCS-EF1α- GreenPuro and delivered as an endo-free mega-scale DNA sample.

3. PCR cloning of the ss-Rluc-C124-N290 from pcDNA-ss-Rluc-C124-N290 template into pCDH-CMVMCS-EF1α-GreenPuro (Ampicillin) via 5’ NheI and 3’ NotI by recombination, with sequence verification across the insert (1016bp); making ss-Rluc-C124-N290 in pCDH-CMV- MCS-EF1α-GreenPuro and delivered as an endo-free mega-scale DNA sample.

4. PCR cloning of the NGLY1 from pcDNA3.1-Hygro-NGLY1 template into EF1α-MCS-T2A-GFP (Ampicillin) via 5’ NheI and 3’ NotI by recombination, with sequence verification across the insert (1985bp); making NGLY1 in EF1α-MCS-T2A-GFP and delivered as an endo-free mega-scale

DNA

sample.

5. PCR cloning of the NGLY1-Del8 from pcDNA3.1-Hygro-NGLY1-Del8 template into EF1α- MCS-T2AGFP (Ampicillin) via 5’ NheI and 3’ NotI by recombination, with sequence verification across the insert (1874bp); making NGLY1-Del8 in EF1α-MCS-T2A-GFP and delivered as an endo-free mega-scale DNA sample.

6. PCR cloning of the ss-Rluc-C124-N290 from pcDNA-ss-Rluc-C124-N290 template into EF1α- MCST2A-GFP (Ampicillin) via 5’ NheI and 3’ NotI by recombination, with sequence verification across the insert (1016bp); making ss-Rluc-C124-N290 in EF1α-MCS-T2A-GFP and delivered as an endo-free mega-scale DNA sample.

7. Lentivirus packaging with 3rd generation packaging system and the above 6 constructs. Ten 25μL aliquots (250μL total) of VSV-G pseudotyped lentiviral particles at >10^9 IFUs/ml titer will be provided, along with titer measurement based on infectious particles.

8. Lentivirus packaging with 3rd generation packaging system and Firefly luciferase control, CMV-rFLuc-T2A-EGFP-mPKG-Puro. Ten 25μL aliquots (250μL total) of VSV-G pseudotyped lentiviral particles at >10^9 IFUs/ml titer will be provided, along with titer measurement based on infectious particles.

9. PCR cloning of the NGLY1 from pcDNA3.1-Hygro-NGLY1 template into pLenti-P2A-tGFP (Chloramphenicol) via 5’ NheI and 3’ NotI by recombination, with sequence verification across the insert (1984bp); making NGLY1 in pLenti-P2A-tGFP and delivered as an endo-free mega-scale DNA sample.

10. PCR cloning of the NGLY1-Del8 from pcDNA3.1-Hygro-NGLY1-Del8 template into pLenti- P2A-tGFP (Chloramphenicol) via 5’ NheI and 3’ NotI by recombination, with sequence verification across the insert (1873bp); making NGLY1-Del8 in pLenti-P2A-tGFP and delivered as an endo-free mega-scale DNA sample.

11. PCR cloning of the ss-Rluc-C124-N290 from pcDNA-ss-Rluc-C124-N290 template into pLenti-P2AtGFP (Chloramphenicol) via 5’ NheI and 3’ NotI by recombination, with sequence verification across the insert (1015bp); making ss-Rluc-C124-N290 in pLenti-P2A-tGFP and delivered as an endo-free megascale DNA sample.

12. PCR cloning of the NGLY1 from pcDNA3.1-Hygro-NGLY1 template into pEF1alpha-IRES- ZsGreen1 (Kanamycin) via 5’ EcoRI and 3’ BamHI by recombination, with sequence verification across the insert (1983bp); making NGLY1 in pEF1alpha-IRES-ZsGreen1 and delivered as an endo-free mega-scale DNA sample.

13. PCR cloning of the NGLY1-Del8 from pcDNA3.1-Hygro-NGLY1-Del8 template into pEF1alpha-IRESZsGreen1(Kanamycin) via 5’ EcoRI and 3’ BamHI by recombination, with sequence verification across the insert (1872bp); making NGLY1-Del8 in pEF1alpha-IRES- ZsGreen1 and delivered as an endo-free mega-scale DNA sample.

14. PCR cloning of the ss-Rluc-C124-N290 from pcDNA-ss-Rluc-C124-N290 template into pEF1alpha-IRES-ZsGreen1 (Kanamycin) via 5’ EcoRI and 3’ BamHI by recombination, with sequence verification across the insert (1014bp); making ss-Rluc-C124-N290 in pEF1alpha- IRES-ZsGreen1 and delivered as an endo-free mega-scale DNA sample.

GOVERNMENT RESPONSIBILITIES

In order to perform custom cloning and lentiviral preparations, we will provide the following materials (empty vectors and vectors containing genes of interest) to the contractor:

1. pcDNA3.1-Hygro-NGLY1

2. pcDNA3.1-Hygro-NGLY1-Del8

3. pcDNA-ss-Rluc-C124-N290

4. pCDH-CMV-MCS-EF1α-GreenPuro

5. pLenti-P2A-tGFP

6. pEF1alpha-IRES-ZsGreen1

DELIVERY OR DELIVERABLES

The Contractor will deliver endo-free mega-scale DNA samples for items 1-12 below. The Contractor will also provide ten 25 μL aliquots (250 uL total) of VSV-G pseudo-typed lentiviral particles at >10^9 IFUs/ml titer, along with titer measurements for items 1-6 and 13 below.

1. NGLY1 in pCDH-CMV-MCS-EF1α-GreenPuro

2. NGLY1-Del8 in pCDH-CMV-MCS-EF1α-GreenPuro

3. ss-Rluc-C124-N290 in pCDH-CMV-MCS-EF1α-GreenPuro

4. NGLY1 in EF1α-MCS-T2A-GFP

5. NGLY1-Del8 in EF1α-MCS-T2A-GFP

6. ss-Rluc-C124-N290 in EF1α-MCS-T2A-GFP

7. NGLY1 in pLenti-P2A-tGFP

8. NGLY1-Del8 in pLenti-P2A-tGFP

9. ss-Rluc-C124-N290 in pLenti-P2A-tGFP

10. NGLY1 in pEF1alpha-IRES-ZsGreen1

11. NGLY1-Del8 in pEF1alpha-IRES-ZsGreen1

12. ss-Rluc-C124-N290 in pEF1alpha-IRES-ZsGreen1

13. CMV-rFLuc-T2A-EGFP-mPKG-Puro

The product will be delivered to NCATS at the following address:

Mitali Tambe Biologist 9800 Medical Center Dr-BLDG C RM 317W Rockville, MD 20850 E-mail: mitali.tambe@nih.gov Phone number: 301-435-2398

The Contractor will deliver the product within 26-40 business days from the date of contract award.

REPORTING REQUIREMENTS

The Contractor will provide the custom cloned DNA constructs and the lentiviral preparations along with a written report stating the concentrations of the final constructs and lentiviral preparations.

OTHER CONSIDERATIONS

Travel:

N/A Key Personnel:

N/A Information System Security Plan:

N/A Data Rights:

The right to all data belongs to NCATS.

Section 508—Electronic and Information Technology Standards:

N/A Publications and Publicity:

N/A Confidentiality of Information:

mailto:mitali.tambe@nih.gov

Any information provided to the contractor by NCATS or collected by the contractor on behalf of NCATS shall be used only for the purpose of carrying out the provisions of this contract and shall not be disclosed or made known in any manner to any persons except as may be necessary in the performance of the contract.