Hematopoietic Stem Cell Transduction for Gene Perturbation
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Solution Overview
Problem
Current techniques for modulating gene expression in immune system cells, such as using retroviruses, face challenges in transducing non-proliferating cells like resting T and B cells, often perturbing the cell state and complicating the interpretation of gene expression modulation experiments.
Innovation Solution
The method involves transducing hematopoietic stem cells with a viral vector that genomically integrates a gene expression perturbation construct, allowing for inducible expression of exogenous nucleic acids like shRNA, and transplanting these cells into immunocompromised animals to reconstitute the immune system, enabling the study of gene function in vivo without altering the natural cellular differentiation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If retroviral transduction is used to modulate gene expression in immune cells, then gene expression can be perturbed, but the cell state is perturbed and natural differentiation is altered
Solution Approach 1:
The patent applies preliminary action by transducing hematopoietic stem cells with the viral vector before transplantation into immunocompromised animals. This allows the gene expression perturbation construct to be integrated into the genome of stem cells in advance, so that when these cells differentiate in vivo, the perturbation occurs naturally during differentiation rather than requiring ex vivo manipulation of differentiated cells
Solution Approach 2:
The patent uses hematopoietic stem cells as an intermediary. Instead of directly transducing differentiated immune cells (which would perturb their state), the viral vector is introduced into stem cells that serve as intermediaries. These transduced stem cells then give rise to differentiated immune cells in vivo, allowing gene expression modulation without directly perturbing the differentiated cell state
2Productivity
If T cells are activated to achieve transduction with viral vectors, then transduction efficiency increases, but cell activation alters the critical differentiation phase
Solution Approach 1:
The patent performs transduction in advance on hematopoietic stem cells before they undergo differentiation. By integrating the gene expression perturbation construct into the stem cell genome beforehand, the invention avoids the need to activate differentiated T cells for transduction, thereby maintaining the natural differentiation process without activation-induced alterations
Solution Approach 2:
The patent uses hematopoietic stem cells as intermediaries to bypass the need for T cell activation. The viral vector is delivered to stem cells which then differentiate in vivo to produce the desired T cell populations, eliminating the requirement to activate T cells for transduction and preserving natural differentiation
3Reliability
If shRNA constructs are delivered to T cells through viral transduction, then gene perturbation can be achieved, but the approach is limited to a small number of candidate genes
Solution Approach 1:
The patent employs a universal viral vector system that can deliver shRNA constructs targeting any candidate gene. By using a versatile lentiviral vector platform that integrates into the genome of hematopoietic stem cells, the system can be adapted to study multiple different candidate genes by simply changing the shRNA sequence, thereby achieving broad candidate gene coverage rather than being limited to a small number of genes
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for efficient and controlled modulation of gene expression in immune cells, preserving their natural state and enabling the study of immune responses and cellular differentiation processes, including the investigation of autoimmune, allergic, and cancer immunotherapy applications.
Implementation Method 1
transducing the cells with at least one viral vector, wherein each viral vector integrates an exogenous nucleic acid into the genome of the cell
Data Source
AI summary
The invention provides methods and compositions for perturbing gene expression in hematopoietic cell lineages in vivo.