Protamine

After bulk TCR sequencing, top rank alpha chains and beta chains were selected and randomly paired to obtain a library of 116 candidate TCR constructs. Detailed information on the library can be found in Table S3. The TCR library was cloned into lentiviral vectors for T‐cell transduction. Multiplicity of infection (MOI) was controlled at 0.2 to ensure single lentivirus integration into the genome of each transduced T cell.
Lentiviruses were generated using the pMD2.G and pSPAX2 envelope plasmids in HEK293T cells. Freshly isolated PBMCs were activated with anti‐CD3 and anti‐CD28 (Miltenyi Biotec) overnight, and proceeded to virus infection. Viral supernatant mixed with 400 U/ml IL‐2 (R&D System, USA) and 8 μg/ml protamine (Sigma Aldrich) was added onto peripheral blood lymphocytes (PBLs) and centrifuged at 800 g and 25°C for 1.5 h. Transduced T cells were maintained in GT551‐H3 medium (TaKaRa) supplied with 5% human serum (GemCell), 1% penicillin/streptomycin (HyClone), and 400 IU/ml IL‐2. The expression of TCR genes was routinely examined using a mouse TCRβ‐specific antibody (BD Biosciences) by FACS.

After determining the presence of HbS in the bulk population, the culture was single-cell cloned. The clones were subjected to qPCR analysis using PCR-based genotyping (Sickle FWD4 primer: 5′-GG CAG AGC CAT CTA TTG CTT AC-3′, Sickle REV2 primer: 5′-CCA ACT TCA TCC ACG TTC ACC-3′, and Sickle probe: 5′-FAM-CTG ACT CCT GTG GAG AA-3′). A selected sHUDEP-2 clone (2 × 10⁵ cells) was pre-stimulated with fresh 1 mL expansion media overnight in a 12-well plate. The culture medium was renewed with a fresh 1 mL expansion medium containing a βT87Q-globin expressing lentiviral vector³³ (link) at different MOIs (0.5, 1, 2, 5, 10, and 25) and protamine (2 mg/mL; Sigma-Aldrich). The next day, transduced cells were split into two wells in 12-well plates in 1 mL fresh expansion media. Transduced cells at different MOIs were single-cell cloned, and genomic DNA from each clone was extracted using an automated DNA system following the manufacturer’s recommendation (Biomek 400; Beckman Coulter, Brea, CA, USA). VCN was determined for each clone by qPCR using the QuantStudio 6 Flex real-time PCR system (Thermo Fisher Scientific) as previously described.³⁴ (link) In addition, globin transcripts per α-globin was calculated as described previously.³⁵ (link)

MDA-MB-231, NRK, and HeLa S3 cell lines were grown according to American Type Culture Collection (Manassas, VA) guidelines. The stable MDA-MB-231 cell lines that overexpress GOLPH3-IRES-GFP, GOLPH3-R90L-IRES-GFP, or IRES-GFP were constructed with the pBABE-Puro retroviral system (Morgenstern and Land, 1990 (link)). Empty retroviral expression vector pBABE-Puro and packaging vectors pUMVC and pVSV-G were kind gifts from Jing Yang (University of California, San Diego). The expression vector of wild-type GOLPH3 was generated by serial cloning to combine GOLPH3 (Dippold et al., 2009 (link)) and IRES-hrGFP (Stratagene/Agilent, Santa Clara, CA) into the BamHI and EcoRI sites of pBABE-Puro vector. Expression and packaging vectors were transfected into HEK293T packaging cells using TransIT-LT1 (Mirus, Madison, WI). Viral supernatants harvested at 48 and 72 h after transfection were filtered through a 0.45 μm filter (Thermo Fisher Scientific, Waltham, MA) and incubated with target MDA-MB-231 cells in the presence of 6 μg/ml protamine (Sigma, St. Louis, MO). 24 h after the second viral infection, 0.5 μg/ml puromycin (InvivoGen, San Diego, CA) was used to select and maintain stable cell pools. The cell lines overexpressing IRES-GFP and R90L-IRES-GFP were generated in similar manner.