Fig 1.
Modeling gene therapy in FA HSCs and oral epithelium.
(A) Hematopoietic stem cell (HSC) gene therapy in FA. FA HSCs (grey) are harvested from FA patient bone marrow, corrected ex vivo (green), then re-implanted to expand in vivo. (B) Corrected HSCs (green) have a proliferative advantage compared to FA HSCs (grey) and outcompete uncorrected HSCs over time in vivo. (C) Oral mucosal gene therapy in FA. In situ gene delivery (here via microneedles) into oral epithelium to correct the underlying FANC gene defect. Over time, corrected cells (green) may or may not clonally expand within FA oral epithelium to replace FA host cells (grey). (D) In our model, FA epithelial cells (light grey) are displaced (light grey with bands) by dividing FA cells (dark grey, top), in contrast to gene-corrected epithelial cells (green) that can resist displacement by dividing neighbors (dark grey) to persist and expand in the basal layer proliferative niche (bottom).
Fig 2.
Mucosal gene therapy depends on a strong proliferative advantage of gene-corrected cells.
(A) 10 gene-corrected cells (green) were tracked over 50 years in a 0.67 mm2 FA mucosal tissue section (100 replicate simulations per condition). The gene correction fates were tracked to identify simulations reaching confluence (80% of basal layer cells corrected, red box) or loss (no remaining gene-corrected cells, blue box). (B) Number of simulations achieving confluence (red), loss (blue), or ongoing expansion (orange) at 50 years as a function of persistence coefficient ( = 0, 0.001, 0.01, 0.2, 0.5, 1). (C) Time at which simulations reached confluence or loss as a function of persistence coefficient, with mean times for each
indicated in black (+) and outcome of individual simulations as points. Gene-corrected patches not reaching confluence or loss by 50 years are not plotted. (D) Number and size of gene-corrected patches at 1 year as a function of persistence coefficient
. (E) Average area of confluent gene-corrected patches as a function of time and persistence coefficient
. Bars indicate interquartile ranges.
Fig 3.
Higher corrected cell numbers and optimal spacing can increase the likelihood of tissue replacement despite clonal interference.
Gene-corrected patch loss probability and time to confluence as a function of corrected cell number (A-C) and spatial delivery distribution (D-F). 100 simulations/condition were run for each persistence coefficient , with error bars indicating binomial errors (B,E) or interquartile ranges (C, F). (A) Initial spatial distribution of gene-corrected cells (
cells, in green) with a constant diffusion coefficient (
). Probability of gene-corrected patch loss (B) and time to confluence (C) as a function of persistence coefficient (
) and initial corrected cell number (
). (D) Initial spatial distribution of ten gene-corrected cells (green) as a function of diffusion coefficient (
, corresponding to low, medium and high diffusion). Probability of gene-corrected patch loss (E) and time to confluence (F) as a function of persistence coefficient (
) and diffusion coefficient (
). Visualizations of gene-corrected cell patches ten years after correction as a function of microneedle spacing (G) or density (H) on 10.67 mm² tissue sections. Initial arrayed delivery shown in panel insets, and each color represents the descendants of a single microneedle that corrects
cells with
. Areas of corrected cell patches at ten years are quantified in the lower right corner of each tissue section.
Fig 4.
Gene correction reduces spread of TP53 mutations through FA tissue.
(A) Model of four cellular genotypes at two loci: FA cells (FANC–/TP53+) in grey; FA cells with TP53 mutations (FANC–/TP53–) in magenta; gene-corrected cells (FANC+/TP53+) in green; gene-corrected cells with TP53 mutations (FANC+/TP53–) in cyan. Arrows indicate transitions that can occur via mutation or gene correction. (B) Genotype-specific persistence coefficients for (A). (C-H) TP53– tissue coverage as a function of gene correction at 46 years in 0.33 mm2 simulated tissue sections, comparing no correction (Ø) to correction with or
. Panels used 100 simulations at a given persistence coefficient except in panels C,F where 300 simulations were performed. C-E show the proportion of tissue coverage at 46 years with (C) comparable TP53 mutation rates and persistence coefficients regardless of FA genotype, (D) an elevated TP53 mutation rate in FANC– cells (
,
) and equivalent TP53 persistence coefficients or (E) equal TP53 mutation rates in FANC– and FANC+ cells with elevated TP53 persistence coefficients in FANC– cells (
). Error bars represent standard errors. Yellow bars represent TP53 tissue coverage range from Martincorena et al. [48] for normal esophagus. (F-H): six representative tissue sections at 46 years from C-E, with colors corresponding to genotypes shown in (A). (G) Consequence of an 8-fold increase in
, and (H) a 4-fold increase in
.