# LV-Barcoding analyses

This repository contains the key scripts used to generate the final tables and figures featured in the accompanying manuscript:  
**Zonari E, Naldini MM, Barcella M, Volpin M, et al**, _Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells from Human Mobilized Peripheral Blood for Gene Therapy Applications_, 2026.

## Abstract 

Ex vivo expansion of mobilized peripheral blood (mPB) hematopoietic stem cells
(HSCs) represents a promising approach to advance cell and gene therapy strategies
yet is hampered by loss of stem cell function when applying commonly used culture protocols. We performed in-depth characterization of mPB expansion cultures by
single cell RNA sequencing, which highlighted differentiation trajectories with
preservation of lineage fidelity in committed progenitors. Defining a putative HSC
cluster allowed an estimation of transduction efficiency in ex vivo cultures, which
correlated with long-term gene marking in xenografts and patients enrolled in a gene
therapy study. We then developed a clinically translatable, GMP-compliant process to
expand lentivirus (LV)-transduced HSCs from mPB of pediatric patients and adult
donors, by biologically informed protocol improvements of cytokine supplementation,
media choice, timing of LV transduction and combinations of small molecules
preventing the activation of differentiation programs. Our optimized process
outperforms validated state-of-the-art cord blood expansion protocols when applied to
mPB. LV integration site analysis and genomic barcode-based clonal tracking provided
definitive proof for symmetric HSC self-renewal divisions occurring during ex vivo
culture. These results warrant clinical testing of this HSC transduction/expansion
process in an upcoming clinical gene therapy trial for autosomal recessive
osteopetrosis (EU CT 2024-518972-30).


## Raw Data

[DP-like_EX-U] :  [PRJEB76976](https://www.ebi.ac.uk/ena/browser/view/PRJEB76976).   
[SCGM] :  [PRJEB77101](https://www.ebi.ac.uk/ena/browser/view/PRJEB77101).  
[TD-Timing] :  [PRJEB77102](https://www.ebi.ac.uk/ena/browser/view/PRJEB77102). 

## Repository structure

In the main folder the main function for processing data produced by Barseq pipeline [Ferrari, Beretta et al, 2021](https://pubmed.ncbi.nlm.nih.gov/34031609/).  Below a glimpse of the file produced:  

| Barcode | Count | SaturationPerc |
| ------ | ------ | -------------- |
| TCTTTACTATAAAAAA | 19689 | 6.80  |
| GGATGTCCCTTATAAT | 17875 | 12.97  |
| TTAGGTTGTAATCATT | 14521 | 17.99  |
| CGGAATAATTATTGGC | 14363 | 22.95  |
| ATATTTACTATAACAC | 13786 | 27.71  |
| TAGGATGAAACATTTG | 13764 | 32.46 |
| TTACTAATTAATCTTA | 13425  | 37.10  |
| TAAATTCATACAATCC | 5939 | 39.15  |
| ACTGATGCTTCACGAA | 3067 | 40.21  |


This file (one for each sample) are then mainly processed by using the custom function [Charting_BCs_Light.R](http://www.bioinfotiget.it/gitlab/custom/zonari_mpbhscexp_2025/zonari_mpbhscexp_2025_barcoding/-/blob/main/ChartingBCs_Light.R) script and futherly analyzed with scripts present in each folder (one script for each experiment):  
 
[DP-like_EX-U](http://www.bioinfotiget.it/gitlab/custom/zonari_mpbhscexp_2025/zonari_mpbhscexp_2025_barcoding/-/tree/main/DP-like_EX-U)  
[SCGM](http://www.bioinfotiget.it/gitlab/custom/zonari_mpbhscexp_2025/zonari_mpbhscexp_2025_barcoding/-/tree/main/SCGM)   
[TD-Timing](http://www.bioinfotiget.it/gitlab/custom/zonari_mpbhscexp_2025/zonari_mpbhscexp_2025_barcoding/-/tree/main/TD-Timing)  

Processed data are stored in Data File 1,4,5 and 6.