Retroviral Vector Pseudotyping for Resting B Cell Transduction
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Solution Overview
Problem
Conventional methods face challenges in efficiently transducing and differentiating B cells, particularly nondividing cells, for gene therapy and immunotherapy applications due to low infectivity of retroviral vectors and difficulty in activating and differentiating B cells in vitro.
Innovation Solution
The method involves transducing resting B cells with retroviral vectors pseudotyped with measles virus glycoproteins H and F, and contacting them with CD40L and B cell activating factors like IL-2, IL-7, IL-10, and CpG to differentiate them into plasma cells that express a nucleic acid of interest, such as antibodies, under conditions that achieve at least 20% transduction efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If conventional retroviral vectors are used to transduce resting B cells, then the method is simple, but the infectivity is low and transduction efficiency is poor
Solution Approach 1:
The patent uses measles virus glycoproteins (H and F) as intermediary pseudotype proteins on the retroviral vector surface. These glycoproteins act as mediators that enable efficient binding and entry into resting B cells, which are normally resistant to conventional retroviral transduction. The pseudotyped vector maintains the simplicity of retroviral delivery while achieving high transduction efficiency through the measles glycoprotein intermediary.
2Productivity
If B cells are activated and differentiated in vitro, then they can produce proteins of interest, but the process takes weeks and is time-consuming
Solution Approach 1:
The patent performs preliminary transduction of resting B cells with the retroviral vector before activation and differentiation. By pre-introducing the gene of interest into the B cell genome while it is in a quiescent state, the subsequent activation and differentiation processes proceed more rapidly. The cell already contains the necessary genetic material, so once activated, it can immediately begin producing the protein of interest, reducing the overall culture time from weeks to days.
3Reliability
If resting B cells are transduced, then transduction efficiency improves, but the cells require complex activation and differentiation conditions
Solution Approach 1:
The patent segments the B cell lifecycle into distinct phases: (1) transduction of resting B cells with the retroviral vector, (2) activation phase using specific stimuli, and (3) differentiation phase leading to plasma cell formation. This segmentation allows each phase to be optimized independently. The resting B cells are transduced under simple conditions, then subsequently activated and differentiated using standardized protocols, achieving high transduction efficiency without requiring complex simultaneous conditions throughout the entire process.
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 the rapid production of proteins of interest, reducing the culture time from weeks to days, providing commercial and safety advantages by effectively differentiating and activating B cells for therapeutic and prophylactic applications.
Implementation Method 1
Antigen is taken up by the B cell through receptor-mediated endocytosis and processed
Implementation Method 2
contacting the transduced resting B cells with a composition comprising CD40L in combination with a B cell activating factor comprising one of more of the factors selected from the group consisting of IL-2, IL-7, IL-10, and CpG; under conditions sufficient to differentiate the transduced B cells into a plasma cell
Data Source
AI summary
The present disclosure provides compositions and methods for transducing B cells. In particular, the present disclosure provides for vectors and methods for transducing resting B cells and methods for differentiating same to express a transgene of interest.