75N95020Q00265_Questions_received.pdf

Solicitation Number: 75N95020Q00265 Title: iNDI-Plus (iNDI+), additional alleles for the iPSC NeuroDegeneration Initiative project

Questions received, followed by the Government’s response to the questions are listed in red font below:

Question 1: For KO: Is it acceptable to introduce early stop codons by NHEJ frame shift?

If so, are compound heterozygous acceptable provided both alleles have early stop codons?

NIH Response: --Yes, but the vendor will need to empirically demonstrate that such stop codons are sufficient to completely abrogate production of each targeted gene (including blocking alternative stop codons and splice isoforms).

Question 2: For revertants, how many of each genotype will be provided (ie. how many of the existing SNPs are homozygous and how many heterozygous)?

NIH Response: --we are only requesting that revertants be made for heterozygous lines. 6 independent clones will be required for each SNP.

Further clarification request for NIH Response to Question 2: the RFQ requests 3 or more independent clones for each revertant in the SOW (specific requirement #1) and in the Base Year Contract (evaluation factor 1 and evaluation factor 3). It is now clear that the revertants are being requested for the heterozygous lines.

NIH Response: -- we are only requesting that revertants be made for heterozygous lines. THREE or more independent clones will be required for each SNP.

Question 3: Request for revertants and Halo tagging of SNVs is for # of clones. Is the requirement for homozygous, heterozygous, specified # of each, whatever is obtained?

NIH Response: --Three clones are requested for halo tagging. Halo tags of heterozygous and homozygous and WT are requested for each variant.

Further clarification request for NIH response to Question 3: We don't understand the answer.

For revertants: Do you want the resultant cell lines to have homozygous WT alleles? If yes, does the Halo tag need to be homozygous as well, and that is the WT tag referred to above?

For Halo tagging SNV variants: How many of the SNVs require a homozygous tag and how many require a heterozygous tag?

NIH Response: For 77 wild-type genes, halo-tag of each gene, two independent heterozygous clones and one independent homozygous clone for each gene(total of three clones for each tagged gene). Revertant lines will be generated from heterozygous mutation harboring lines from the ongoing iNDI project, and the mutant allele will be reverted back to a wild-type allele, resulting in a homozygous wild-type genotype. Halo tag lines will NOT be generated from revertant lines. These are independent projects.

For up to 130 SNV heterozygous variants (chosen from the above 77 wild-type genes that are being generated through the original iNDI project). Heterozygous halo-tag for the variant harboring allele only (i.e. not the wild-type allele). Two independent clones for each variant.

Question 4: We assume the revertants are the SNVs made in the first iNDI round. Please confirm.

NIH Response: --This is correct.

Question 5: What is the expectation for molecular confirmation that SNV allele is tagged? Sanger, NGS, something else?

NIH Response: --Sanger sequencing of the entire tag and junctions. Future assays will include pacbio long read sequencing that spans the integration site, and these could be used in place of sanger sequencing once available.

Question 6: Will the government provide sterility data on cells they will provide, or will we need to send them for testing?

NIH Response: --We will provide sterility data for all clones provided to the vendor.

Question 7: Will all projects (revertant, KO, Halo tagging) be awarded to a single contractor, or will it be divided amongst multiple contractors as was the case for the first INDI project? Estimating how we will need to allocate resources to accommodate the project.

NIH response: Single contractor

Question 8: Will all 130 revertants, 77 KO’s, 77 WT Halo tags, 130 SNV Halo tag targets per project be awarded to a single contractor. Or split across multiple contractors?

NIH response: Single contractor

Question 9: Are the non edited parental lines requested as a deliverable for each cell line?

NIH response: NO

Question 10: How will the plasmids be delivered for Halo tagging? Ie. What quantity, what format, what plasmid backbone?

NIH response: Donor plasmids will be synthesized by a third party, provided in maxi-prep format (at least 10ug) with 300-1000bp homology arms on each side of the halotag, in plasmid backbone TBD, chosen based on vendor who synthesizes donor

Question 11: We are not clear on the zygosity requirements.

NIH response: Clarification provided above

Question 12: In what format will the government send the SNV clones that need to be reverted (matrix plates, vials, tubes)?

NIH response: Tubes

Question 13: Will you confirm that all of these edits are to be done in the KOLF2.1 cells as in the first INDI project?

NIH response: KOLF2.1

Question 14: What is the priority of the projects (revertant, KO, Halo tags)?

NIH response: revertant, KO, halo tags, in this order

Question 15: How many cells per vial are required?

NIH response: 1 million cells per vial

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