Determination of functional, physical, and genomic titers

Functional titer was determined by mixing vector with 2 × 10⁴ HEK293Tsa cells in a total volume of 200 μL per well of a 96-well plate and incubated at 37°C, 5% CO₂, for 3–4 days. The percent of cells expressing GFP, determined by flow cytometry using an Accuri C6 (Becton Dickinson) using the gating strategy shown in Figure S1, was multiplied by the starting cell number and normalized to 1 mL of vector supernatant. Units are HEK293Tsa transducing units per mL (TU/mL).

The physical titer was determined using a SYBR-Green product-enhanced reverse transcriptase (SG-PERT) assay according to a published protocol,32 but with the following reagents: PowerUp Sybr Green Master Mix (Thermo Fisher Scientific) and recombinant HIV-1 RT supplied by Abcam.

Genomic titer was determined by extracting RNA from 50 μL of cell-culture medium using a Purelink pro 96 viral RNA/DNA purification kit (Thermo Fisher) according to the manufacturer’s protocol, including the optional on-column DNase step using PureLink DNase set (Thermo Fisher Scientific) and eluting in 100 μL of water. Superscript IV VILO master mix was used to prepare cDNA (Thermo Fisher Scientific). Absolute quantification of the recovered viral genomes per mL was performed by digital droplet PCR (ddPCR) using published primer/probe sequences spanning the MSD33 in conjunction with Bio-Rad QX200 automated droplet generator and droplet reader using ddPCR supermix for probes (Bio-Rad). The absolute copy number was normalized to mL of starting material in the final ddPCR volume. Although recovery efficiency is not known, it was found to be consistent for samples processed in parallel (not shown). All samples within an experiment were processed in parallel.