Novel immunogen-specific variable lymphocyte receptors for creation of chimeric antigen receptors and methods for improved transduction and gene delivery 公开
Moot, Robert (Fall 2017)
Abstract
Gene therapy, as an approach to disease treatment, has applications in a wide variety of prospective fields. This methodology is not without limits, however, as there is still a need to improve both the transduction process as well as the impact of the transgene conveyed via the viral vector. The studies herein focus on improving both transduction efficiency and the functionality of a therapeutic transgene.
The application of the transgene improvement focuses on the use of chimeric antigen receptors (CARs). CARs are a type of engineered, recombinant cell surface receptor that can be expressed on an effector cell to allow for directed recognition of a target antigen. One of the limitations of CARs as a cancer therapy is the availability of tumor cell target antigens. Increasing the number and variety of targetable tumor related antigens would improve its application to a wider variety of tumors. As a way to expand the antigen repertoire to which a CAR can be directed, we have implemented the use of the lamprey variable lymphocyte receptor (VLR). The VLR is the primary component of the adaptive immune system of the lamprey and hagfish. Due to its unique structure, the VLR binds antigen in a fundamentally different manner than traditional immunoglobulin-based antibodies. In addition to an alternate binding geometry, the VLR is also less subject to the constraints of self-tolerance. Combined, these two factors may improve the identification and utilization of cancer cell target antigens.
Along with improving the efficacy of CAR therapy, additional research completed herein has focused on improving the efficiency of transduction with viral vectors. We have identified several methods that may improve transduction efficiency. These include pretreatment of target cells with a small molecule selected from a high-throughput screen, increasing the concentration of virus on the target cells through use of a microfluidics device, and the use of an AAV6 vector to transfer a CAR transgene to innate cells. Together, these techniques may increase the impact of gene therapy by increasing the efficiency of viral gene transfer.
Table of Contents
Chapter 1 – Introduction.................................................................................................. 1
1.1 The history of gene therapy............................................................................... 2
1.2 Viral vectors in gene therapy.............................................................................. 7
A. Lentiviral vector background........................................................................ 7
B. Engineering lentiviral vectors for gene therapy........................................... 12
C. Background and structure of AAV vectors................................................... 14
D. Engineering AAV vectors for gene therapy................................................. 15
1.3 Immunotherapy background.............................................................................. 16
A. Cancer Immunotherapy overview................................................................ 19
B. Monoclonal antibodies................................................................................. 20
C. Immune checkpoint inhibitors..................................................................... 21
D. Chimeric antigen receptors overview............................................................ 22
E. Clinical trials with chimeric antigen receptors............................................... 24
1.4 Overcoming the limitations in tumor cell targeting.......................................... 25
A. Variable lymphocyte receptors.................................................................... 26
Chapter 2 – Development and screening of immunogen specific VLRs against FVIII and the neuroblastoma cell line SK-N-Be(2) 33
2.1 Abstract............................................................................................................ 34
2.2 Introduction...................................................................................................... 34
2.3 Materials and Methods...................................................................................... 39
2.4 Results.............................................................................................................. 45
2.5 Discussion......................................................................................................... 64
Chapter 3 – The use of VLRs as the antigen recognition region of chimeric antigen receptors 68
3.1 Abstract............................................................................................................ 69
3.2 Introduction...................................................................................................... 69
3.3 Materials and Methods...................................................................................... 71
3.4 Results.............................................................................................................. 75
3.5 Discussion......................................................................................................... 97
3.7 Supplemental Information.............................................................................. 100
Chapter 4 – Increasing transduction efficiency........................................................ 107
3.1 Abstract.......................................................................................................... 108
3.2 Introduction.................................................................................................... 109
3.3 Materials and Methods.................................................................................... 114
3.4 Results............................................................................................................ 122
3.5 Discussion....................................................................................................... 146
3.7 Supplemental Information.............................................................................. 152
Chapter 5 –Discussion and future directions............................................................ 153
4.1 Discussion....................................................................................................... 154
4.2 Results and implications.................................................................................. 155
4.3 Future Directions............................................................................................ 162
4.4 Conclusions..................................................................................................... 162
References...................................................................................................................... 165
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