GENETICALLY MODIFIED ERAP 2 N AND ITS EFFECT

GENETICALLY MODIFIED ERAP 2 N AND ITS EFFECT ON LYMPHOCYTE ACTIVATION Shradda Adhikari Mentor: Eun D. Lee

Spontaneous Miscarriages ■ Most common type of pregnancy loss- more than 3 billion cases per year in the US ■ Possible reason: immune system imbalance ■ TNF= Tumor neurosis Factors- induced by the lymphocytes that is known to be a factor in repeated miscarriages (1) – NKCs (CD 16 and CD 56) (2)

ERAP 2 and the Immune System ■ White blood cells (lymphocytes) work by detecting antigens on cells ■ ERAP 2 cleaves these antigens to be presented on HLA-C 1 Figure from Ref. 5

ERAP 2 N and ERAP 2 K ■ N 392 K (asparagine and Lysine) (4) ■ Determined by SNPs ■ ERAP 2 N has 165 times the cleaving capability (4) Figure: the cleaving efficiency of ERAP 2 N vs ERAP 2 K

Genetics of ERAP 2 N and K ■ ERAP 2 N IS NOT FOUND IN NATURE ■ Figure: Haplotype A (ERAP 2 K) and Haplotype B (no protein) ■ B: SNP rs 2248374 G and SNP rs 2549782 is A ■ Chilean population: not linked, but STILL didn’t express the N allele (6)

Haplotype A: …ATGATATTTGGCTTAA GAGGG… rs 2549782 G …TGACAAGTAACATGGT GG… rs 2248374 392 ERAP 2 K … Lysine … AA

Haplotype B: …ATGATATTTGGCTTAA AGGG… rs 2549782 TG …TGACAAGTAACATGGT AGG… rs 2248374 NO FUNCTIONING G

Central Question Is ERAP 2 N possible in nature by a single nucleotide mutation? And if so, will it cause a dramatic difference in immune system activity?

Goal …TGACAAGTAACATGGT AGG… …ATGATATTTGGCTTAA AGGG… rs 2549782 TG …TGACAAGTAACATGGT GG… rs 2248374 …CTT - AAT G AA - GAG… 392 ERAP 2 N … Asparagine … Immune response

Parts of the Experiment ■ 1. Gene editing – a. transfection crispr cas 9 – b. transfection target DNA homologous recombination – C. western blot ■ 2. Lymphocyte activation assay – a. Isolation of lymphocytes – b. Insertion of antibodies – c. Quantification of activated lymphocytes

Genetic Modification JEG-3 Cells: Homozygous for haplotype B (BB) Target: change rs 254978 G--> A so the N allele can be translated Crispr Cas 9 editing: ■ cr. RNA ■ g. RNA ■ PAM 5’-NGG-3’

What is the cr. RNA, g. RNA, PAM? ■ cr. RNA: 20 nucleotide RNA sequence that can bind to the target sequence 5’AAGATGACAAGTAACATGGTGAGG -3’ green= cr. RNA, blue= g. RNA. The PAM sequence is pointed out

Plasmid Transcribes into cr. RNA, SNP rs 2248374 ORIgene® plasmid kit

Transfection

cr. RNA binds to target sequence and Cas 9 cleaves the target sequence

Donor ds. DNA transfection Donor DNA: …ATCTGGTGGAGTTTGTCATTCGGATCCCAAGATGACAAGT A AACATGGT AGGATAAAGAGAGTCACAGAGTAGAAGAGAT CTGTGGAAT…

Homologous recombination

Goal …TGACAAGTAACATGGT AGG… …ATGATATTTGGCTTAA AGGG… rs 2549782 TG …TGACAAGTAACATGGT GG… rs 2248374 …CTT - AAT … Asparagine AA - GAG… LAA 392 ERAP 2 N G … Immune response

Lymphocyte Activation Assay 4 , D C 8, CD 16 D C 56, D C 69 CD NKC s, T- cells Fluorescence tagged

Quantification by Flow cytometry ■ Cells are washed and the lymphocytes are put through flow cytometry ■ Can tell how much of each antibody in each lymphocyte ■ CD 16, CD 56= NKCs CD 4, CD 8= T cells CD 16= early activation

Results ■ 1. The gene editing works and ERAP 2 N is transcribed ■ 2. ERAP 2 N cleaves more peptides and induces a greater immune response

Improvement/Pitfalls ■ Homologous repair is not as efficient by non homologous end joining ■ Antibody binding in the solution

THANK YOU Questions?