Recombinant Adeno-Associated Virus

Editing the genome without breaking it.

Universal Cells uses recombinant adeno-associated virus (rAAV)-mediated gene editing to efficiently edit chromosomal genes without the use of genotoxic nucleases. rAAV vectors are effective and safe, and have been used in numerous clinical trials. We have licensed a stem cell-tropic rAAV vector serotype for engineering human pluripotent stem cells. Our technology allows us to produce customized stem cells that contain deletions, insertions, or point mutations at any genomic position.

Unlike nuclease-based genome editing, our approach is not genotoxic. It does not require a double strand break, generate off-target alterations to the genome, or produce unwanted mutations at the target site. It also does not introduce nuclease genes into the cell that may have unintended effects.

rAAV-Mediated Gene Editing

Universal Donor Cells

No immune suppression.  No donor matching.  No rejection.

Universal donor cell strategy diagram

A major advantage of universal donor cells is that a single cell line can be differentiated into a therapeutic cell type, tested for regulatory approval, and used in multiple recipients, creating an off-the-shelf product. We can provide our engineered stem cells, or our gene editing technology can be applied to a stem cell line of choice to create customized donor cells.

Universal Cells addresses the allogeneic rejection problem by manipulating human leukocyte antigen (HLA) expression in human stem cells. Multiple HLA class I and class II proteins must be matched for histocompatibility in allogeneic recipients. We eliminate the expression of these polymorphic HLA proteins by gene editing, express specific non-polymorphic HLA molecules in order to provide essential class I signals that block lysis by Natural Killer cells, and introduce suicide genes for enhanced safety.

Significant evidence supports the concept that these HLA-engineered cells will function normally and avoid allogeneic rejection after transplantation.

1) Rare individuals who are HLA class I-negative or HLA class II-negative are relatively healthy, demonstrating that HLA-negative human cells can form all essential organs.

2) Mice lacking major histocompatibility complex (MHC) class I and class II antigens (the murine equivalent of HLA) have been extensively studied and are healthy except for a lack of CD4+ and CD8+ T cells. Importantly, transplantation experiments have shown that organs or cells from class I-negative mouse donors survive longer in allogeneic recipients (sometimes persisting indefinitely), including liver cells, kidneys, hearts and pancreatic islets. These mouse experiments suggest that HLA-engineered human cells will also survive longer than allogeneic cells in many of the clinical settings being considered for pluripotent stem cells.

HLA class I engineering

HLA-A, B and C are polymorphic class I proteins expressed by most nucleated cells that must be matched for histocompatibility. The Beta-2 Microglobulin (B2M) gene encodes a common subunit essential for cell surface expression of all HLA class I heterodimers (the other subunits are the heavy chains for HLA-A, B, C, E, F, or G), so B2M-/- cells are class I-deficient. We edit both B2M genes to create human pluripotent cells that lack polymorphic class I proteins. These editing steps also introduce a Thymidine Kinase (TK) suicide gene to allow for in vivo elimination of transplanted cells.

For some cell types, a lack of class I expression leads to lysis by Natural Killer (NK) cells. To overcome this “missing self” response, we simultaneously knock in the heavy chain of the non-polymorphic HLA-E gene using HLA single chain technology (see below). This provides a class I-positive signal to inhibit NK cells. These class I-engineered stem cells can serve as universal donor cells in applications where the differentiated cell product does not express HLA class II.

Class I strategy

Introducing a Single Chain HLA Gene

 

HLA class II engineering

Unlike HLA class I, class II proteins lack a common subunit that can be edited to prevent surface expression. Therefore our approach is to edit both copies of the RFXANK transcription factor gene required for class II expression. Patients with RFXANK mutations do not express HLA class II molecules on their antigen-presenting cells. Combining class I and class II engineering creates a universal donor stem cell line appropriate for deriving any differentiated cell product.

If desired, specific HLA class II alpha and beta chain transgenes can be expressed in RFXANK-/-cells from a RFXANK-independent promoter.

Class II strategy

Transcription Factors Controlling Class II Expression