Vector Copy Distribution at a Single-Cell Level Enhances Analytical Characterization of Gene-Modified Cell Therapies

The ability to deliver transgenes into the human genome using viral vectors is a major enabler of the gene-modified cell therapy field. However, the control of viral transduction is difficult and can lead to product heterogeneity, impacting efficacy and safety, as well as increasing the risk of batch failure during manufacturing. To address this, we generated a novel analytical method to measure vector copy distribution at the single-cell level in a gene-modified, lentiviral-based immunotherapy model. As the limited amount of genomic DNA in a single cell hinders reliable quantification, we implemented a preamplification strategy on selected lentiviral and human gene targets in isolated live single cells, followed by quantification of amplified material by droplet digital PCR. Using a bespoke probability framework based on Bayesian statistics, we show that we can estimate vector copy number (VCN) integers with maximum likelihood scores. Notably, single-cell data are consistent with population analysis and also provide an overall measurement of transduction efficiency by discriminating transduced (VCN ≥ 1) from nontransduced (VCN = 0) cells. The ability to characterize cell-to-cell variability provides a powerful high-resolution approach for product characterization, which could ultimately allow improved control over product quality and safety.

[1]  Fan Shen,et al.  Next generation digital PCR measurement of hepatitis B virus copy number in formalin-fixed paraffin-embedded hepatocellular carcinoma tissue. , 2015, Clinical chemistry.

[2]  S. Bergelson,et al.  Determination of Lentiviral Infectious Titer by a Novel Droplet Digital PCR Method. , 2018, Human gene therapy methods.

[3]  B. Fehse,et al.  Pois(s)on – It's a Question of Dose… , 2004, Gene Therapy.

[4]  David V Schaffer,et al.  Viral Vectors for Gene Therapy: Translational and Clinical Outlook. , 2015, Annual review of biomedical engineering.

[5]  C. Schneider,et al.  Development of the First World Health Organization Lentiviral Vector Standard: Toward the Production Control and Standardization of Lentivirus-Based Gene Therapy Products , 2017, Human gene therapy methods.

[6]  Alexey Bersenev,et al.  Management of ‘out of specification’ commercial autologous CAR-T cell products , 2018, Cell and Gene Therapy Insights.

[7]  Michael L Kaufman,et al.  Gene Therapy for Sickle Cell Disease: A Lentiviral Vector Comparison Study. , 2018, Human gene therapy.

[8]  H. Nakauchi,et al.  Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy , 2016, Human gene therapy methods.

[9]  Tracy T L Yu,et al.  Recent Progress in European Advanced Therapy Medicinal Products and Beyond , 2018, Front. Bioeng. Biotechnol..

[10]  J. Hoekman,et al.  A decade of marketing approval of gene and cell-based therapies in the United States, European Union and Japan: An evaluation of regulatory decision-making. , 2018, Cytotherapy.

[11]  Robert J. Weatheritt,et al.  Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors. , 2017, Molecular cell.

[12]  Vanessa M. Peterson,et al.  Multiplexed quantification of proteins and transcripts in single cells , 2017, Nature Biotechnology.

[13]  P. Kantoff,et al.  Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Michel Sadelain,et al.  Manufacturing Validation of Biologically Functional T Cells Targeted to CD19 Antigen for Autologous Adoptive Cell Therapy , 2009, Journal of immunotherapy.

[15]  M. Yamada,et al.  Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy , 2017, Molecular therapy. Methods & clinical development.

[16]  Michael Rothe,et al.  Gene Therapy for Wiskott-Aldrich Syndrome—Long-Term Efficacy and Genotoxicity , 2014, Science Translational Medicine.

[17]  H. Swerdlow,et al.  Large-scale simultaneous measurement of epitopes and transcriptomes in single cells , 2017, Nature Methods.

[18]  John R. Haliburton,et al.  Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding , 2017, Scientific Reports.

[19]  Jeff Mellen,et al.  High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number , 2011, Analytical chemistry.

[20]  G. Sanguinetti,et al.  Single cell multi-omics profiling reveals a hierarchical epigenetic landscape during mammalian germ layer specification , 2019, bioRxiv.

[21]  William Stafford Noble,et al.  High-Throughput Single-Cell Sequencing with Linear Amplification. , 2019, Molecular cell.

[22]  J. Perea,et al.  Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction , 2010, Gene Therapy.

[23]  Andrew C. Adey,et al.  Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing , 2015, Science.

[24]  Hendrik Emons,et al.  Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material , 2016, Biomolecular detection and quantification.

[25]  Christof von Kalle,et al.  Side effects of retroviral gene transfer into hematopoietic stem cells. , 2003, Blood.

[26]  Ira M. Hall,et al.  Mosaic Copy Number Variation in Human Neurons , 2013, Science.

[27]  Guideline on quality, non-clinical and clinical aspects of medicinal products containing genetically modified cells , 2012 .

[28]  K. Ellem,et al.  The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events , 2004, Genetic vaccines and therapy.

[29]  G. Nolan,et al.  Mass Cytometry: Single Cells, Many Features , 2016, Cell.

[30]  A. Saliba,et al.  Single-cell RNA-seq: advances and future challenges , 2014, Nucleic acids research.

[31]  Christine Kinnon,et al.  Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. , 2008, The Journal of clinical investigation.

[32]  J. Lee,et al.  Single-cell RNA sequencing technologies and bioinformatics pipelines , 2018, Experimental & Molecular Medicine.

[33]  A. Scott,et al.  Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia. , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[34]  A. Thrasher,et al.  Treating Immunodeficiency through HSC Gene Therapy. , 2016, Trends in molecular medicine.

[35]  Douglas D. Richman,et al.  Highly Precise Measurement of HIV DNA by Droplet Digital PCR , 2013, PloS one.

[36]  F. Bushman,et al.  Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. , 2008, The Journal of clinical investigation.

[37]  G. Cossu,et al.  The committee for advanced therapies' of the European Medicines Agency reflection paper on management of clinical risks deriving from insertional mutagenesis. , 2013, Human gene therapy. Clinical development.

[38]  Benjamin J. Hindson,et al.  Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification , 2011, Analytical chemistry.

[39]  I. Verma,et al.  Lentiviral vectors, two decades later , 2016, Science.

[40]  G. Helenius,et al.  Digital droplet PCR (ddPCR) for the detection and quantification of HPV 16, 18, 33 and 45 - a short report , 2017, Cellular Oncology.

[41]  D. Trono,et al.  A Third-Generation Lentivirus Vector with a Conditional Packaging System , 1998, Journal of Virology.

[42]  William Stafford Noble,et al.  Massively multiplex single-cell Hi-C , 2016, Nature Methods.

[43]  A. Zander,et al.  Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. , 2003, Blood.

[44]  M. Sabatino,et al.  Overcoming Challenges for Engineered Autologous T Cell Therapies , 2018 .

[45]  Steven Jacobson,et al.  Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations , 2014, Journal of NeuroVirology.

[46]  M. Sadelain.  Insertional oncogenesis in gene therapy: how much of a risk? , 2004, Gene Therapy.

[47]  Claudia Zylberberg,et al.  Sources of Variability in Manufacturing of Cell Therapeutics , 2019, Regenerative Engineering and Translational Medicine.