Publications

Synthetic oligodeoxynucleotides (ODNs) have been used as repair templates in gene-editing applications to insert transgenic sequences into defined genomic loci, albeit with low efficiency. Cells engineered in this way are of interest for many therapeutic applications, including allogeneic NK and T cells engineered to express stealthing proteins, cytokines, and chimeric antigen receptors (CARs) for the treatment of a variety of cancers. To increase the efficiency of integration, gene-editing proteins can be co-expressed to create a double-strand break at the target locus. However, recognition of dsODNs by pattern recognition receptors activates signaling cascades resulting in the production of cytokines, including type I interferons such as IFIT1-3 and IFN-β. This immune response can lead to cell cycle arrest, differentiation, and apoptosis and may contribute to low insertion efficiency observed in primary and iPS cells. It has been shown in human immune cells that co-delivery of a short ODN comprising the immunosuppressive motif, TTAGGG, which is found in mammalian telomeric DNA, inhibits the activation of the damage-associate molecular pattern (DAMP) pathway in response to cytosolic DNA. This ODN competitively binds to inflammasomes, and reduces the secretion of proinflammatory cytokines. We hypothesized that the presence of the TTAGGG motif would decrease dsODN-related activation of a pro-inflammatory response in human cells, leading to higher transgene insertion efficiency. We incorporated the TTAGGG motif either at the 5’ end of dsODNs, or delivered it separately on a short single-stranded ODN (A151). Human primary fibroblasts, iMSCs and iPSCs were electroporated with a dsODN encoding a GFP reporter and containing an SfoI restriction site. Upregulation of pro-inflammatory markers including IFIT1-3, was measured by RT-PCR. We observed 29-fold higher expression of IFIT1 and IFIT3 in cells electroporated with dsODNs than in untreated controls. Interestingly, including TTAGGG motifs at the 5’-ends of the dsODNs limited the upregulation of IFIT1 and IFIT3 to 10- and 15-fold, respectively, while co-delivery of the TTAGGG motif prevented their upregulation altogether. We then used a gene-editing endonuclease targeting the AAVS1 safe-harbor locus on chromosome 19 to investigate the impact of the TTAGGG motif on the insertion of transgenes at this site. The TTAGGG motif (whether incorporated in the dsODN or co-transfected in the form of the A151 ODN) resulted in approximately 50% higher viability and approximately 50% more GFP-positive cells than when the motif was not present. We show that immunosuppressive sequences can increase ODN insertion efficiency and improve cell viability, and may therefore be a powerful tool for therapeutic knock-in applications, including the generation of knock-in iPS cell lines.

Cytotoxic lymphocytes, including T cells and NK cells, are being developed as allogeneic, “off-the-shelf”, cell therapies for the treatment of hematological and solid tumors. Allogenic lymphocyte therapies face challenges, however, including limited expansion potential and limited in vivo persistence due to host immune rejection. To address these challenges, we developed an mRNA-reprogrammed iPSC line with a biallelic knockout of the beta-2 microglobulin (B2M) gene, a key component of MHC class I molecules, using an mRNA-encoded chromatin context-sensitive gene-editing endonuclease. We differentiated these B2M-knockout iPSCs using a novel, fully suspension process that replaces specialized micropatterned culture vessels with a spheroid culture step. The resulting lymphocytes were characterized for surface markers via flow cytometry and incubated with cancer cells to assess tumor cell engagement and cytotoxicity. Notably, we observed consistently higher yields of lymphocytes from the B2M-knockout iPSC line than from the parental wild-type iPSC line. Both wild-type and B2M-knockout lymphocytes cells killed 75-90% of K562 cells after 24 hours (effector to target (E:T) ratio of 5:1). Interestingly, cytotoxic lymphocytes derived from B2M-knockout iPSCs exhibited greater K562 cell killing with the addition of IL15 and IL2, while killing by wild-type cells was not controlled by these activating cytokines. Cancer cell killing activity was maintained through cryopreservation, albeit at a reduced level (15-40% reduction in activity). These results suggest that B2M-knockout iPSCs may serve as an ideal source of cytotoxic lymphocytes for the development of “off-the-shelf” allogeneic cell therapies for the treatment of cancer.

Cancer immunotherapy has advanced rapidly over the past two decades, with several autologous chimeric antigen receptor (CAR)-T cell therapies approved for the treatment of hematologic cancers. However, CAR-T cells have shown limited activity against solid tumors, in part due to the immunosuppressive nature of the tumor microenvironment preventing CAR-T cell infiltration. This has led to investigation of other immune cells as alternatives to T-cell-based therapies, including monocytes and monocyte-derived macrophages, which exhibit innate tumor-infiltration properties. We developed a process for differentiating pluripotent stem cells along a myeloid lineage, and generated populations of cells with characteristics of monocytes and M1 and M2 macrophages, including cytokine secretion and tumor cell-killing activity. mRNA-reprogrammed human induced pluripotent stem cells (iPSCs) were differentiated into monocytes using a 28-day monolayer protocol. Beginning on day 14, cells were harvested every 3-4 days. CD14+ isolation yielded >95% CD14+ cells with an average yield of 4.1x10⁴ cells per cm² per harvest. iPSC-derived monocytes were compared to peripheral blood mononuclear cell (PBMC)-derived monocytes for expression of key hematopoietic and myeloid-lineage markers CD11b, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα. iPSC-derived monocytes showed similar expression of CD11b, CD14, CD33, CD45, and CD163 compared to PBMC-derived monocytes, and increased expression of markers indicative of an activated state: CD80 and CD206. Compared to PBMC-derived monocytes, iPSC-derived monocytes showed both higher viability in culture and superior recovery from cryopreservation. iPSC-derived monocytes were further differentiated into macrophages by exposure to MCSF for 3-4 days, and were assessed for their ability to polarize, secrete pro- and anti-inflammatory cytokines, and for cytotoxic activity when co-cultured with cancer cells. M1 macrophages were polarized with interferon gamma (IFN-γ, 50 ng/mL) and lipopolysaccharide (LPS, 10 ng/mL) for 48 hours, while M2 macrophages were treated with IL-4 (10 ng/mL) for 48 hours. iPSC-derived monocytes differentiated into macrophages with >90% efficacy, as assessed by cell adherence, morphology, and surface marker expression (CD14, CD45, CD163). M1 and M2 polarized iPSC-derived macrophages secreted similar levels of TNFα, IL-12p70, and IL-10 compared to PBMC-derived macrophages. iPSC-derived macrophages killed 45% of U2OS cancer cells in vitro after 24 hours at an E:T ratio of 5:1. We demonstrate a process for differentiating mRNA-reprogrammed iPSCs into cytotoxic macrophages. The mRNA reprogramming and differentiation processes are virus-free and DNA-free, avoiding any potential risk of vector integration. These results suggest that mRNA-reprogrammed iPSCs may represent a viable source of macrophages for the development of therapies to treat various indications, including solid tumors.

Gene editing technology, which enables the precision modification of DNA in living cells, is being developed for the treatment of various diseases, including genetic diseases and cancer. Gene editing commonly employs sequence-specific endonucleases to create double strand breaks in genomic DNA, and relies on the cell’s DNA repair mechanisms to apply the desired changes. Precise sequence modifications, such as single-base changes, rely on the homology directed repair (HDR) mechanism. Despite its essential role in gene repair, HDR occurs at a very low frequency in many cells compared to other repair mechanisms. Here, we evaluate the impact of resveratrol, a small molecule extracted from grape skin that has recently been shown to promote the expression of key HDR factors and induce cell cycle arrest at S phase in porcine fetal fibroblasts, on single-base editing efficiency in primary human fibroblasts. Following treatment with resveratrol, fibroblasts were co-transfected with mRNA encoding a chromatin context-sensitive gene-editing protein targeting the AAVS1 safe-harbor locus and a single-stranded DNA repair template designed to introduce a SfoI restriction-enzyme site through a G-to-C mutation. Single-base editing efficiency was determined by restriction fragment length polymorphism (RFLP) analysis. Resveratrol treatment prior to transfection increased the S and G2-phase population 2.3-fold and increased HDR efficiency 2-fold compared to untreated cells. Application of resveratrol after transfection (i.e., no cell cycle synchronization) yielded further improvement in single-base editing efficiency (> 2-fold), suggesting that the effects of resveratrol on HDR are not confined to cell-cycle control. Resveratrol treatment provides a straightforward method for improving HDR efficiency in primary human fibroblasts, and may serve as a useful tool in the development of HDR-based gene-editing therapies.

Induced pluripotent stem cells (iPSCs) have emerged as an exciting platform for developing personalized cell therapies. However, clinical applications are limited by the low efficiency of viral and episomal reprogramming methods and safety concerns related to vector integration and genomic scarring. Using messenger RNA (mRNA) to reprogram cells avoids these issues, however previously reported mRNA-based reprogramming methods required lengthy transfection schedules and suffer from the need to use proprietary media formulations. Here we report a seven-day, high-efficiency, immunosuppressant-free mRNA reprogramming protocol for dedifferentiating adult human dermal fibroblasts into iPSCs. To achieve this, we designed a chemically defined, animal component-free and xeno-free reprogramming medium that contains specially treated albumin, which we show enhances transfection efficiency and supports efficient reprogramming. We measured reprogramming efficiency by counting the number of SSEA4 positive colonies generated from a starting culture of 1x10³ adult human dermal fibroblasts. Using this approach, we measured a 6% reprogramming efficiency, which is a 60-fold improvement to the <0.1% efficiency typically obtained with viral and episomal reprogramming methods. We analyzed iPSCs for expression of the pluripotency markers OCT4 (POU5F1), SSEA4, and NANOG, as well as by differentiating the cells in vitro into mesenchymal stem cells, hematopoietic stem cells, osteoblasts, adipocytes, chondrocytes, T cells, natural killer (NK) cells, cardiomyocytes, and neurons. We also explored the role of other media components, including TGF-β, in mRNA reprogramming and evaluated protein expression, immunogenicity, and reprogramming efficiency of various novel mRNA chemistries and molecular designs.

Sequence-specific gene-editing endonucleases, such as zinc finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9), and transcription activator-like effector nucleases (TALENs) are being used in the development of many gene and cell therapies. However, these gene-editing endonucleases have seen limited in vivo application due to concerns about effects on genomic loci outside the intended cut site. TALENs consist of a DNA binding domain containing repeat-variable diresidues (RVDs) that confer site specificity fused to a nuclease catalytic domain. The RVDs contact individual bases in the target DNA sequence, and are connected by two alpha-helices linked by a short, disordered loop. We hypothesized that engineered gene-editing endonucleases comprising DNA-binding domains containing novel loops could exhibit different on-target cutting activity as well as altered specificity, due to the potential of this linkage to determine the degree of conformational disorder of the DNA-binding domain. We tested the ability of gene-editing endonucleases containing novel linkages to edit genes in primary cells, and discovered a striking temperature-dependence of gene-editing with certain linkages. We identified engineered endonucleases that efficiently edit only at sub-physiological temperatures, as well as endonucleases capable of high-efficiency editing in primary cells at 37 °C. We observed high-efficiency gene editing with the novel endonucleases in primary human dermal fibroblasts, primary epidermal keratinocytes, induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs). Additionally, we measured the specificity of the engineered endonucleases at various editing temperatures in cultured cells using LAM-PCR based sequencing. We show that engineering the RVD linkage of gene-editing endonucleases has the potential to improve on-target activity as well as sequence specificity in gene therapies.

Messenger RNA (mRNA) has many applications in biological research and therapeutic development. However, its relatively short half-life limits the use of mRNA in applications where long-term protein translation is desired. Circular RNAs (circRNAs) are a novel class of RNA molecules with enhanced stability that exist in many organisms, including C. elegans, Drosophila melanogaster, mice, and humans. Engineered circRNAs have been used for a variety of applications including microRNA sponges and protein expression. The covalently closed structure of circRNAs makes them resistant to degradation by exonucleases, leading to extended protein translation in cells. Methods for synthesizing circRNAs include the use of a splint molecule to bring the 5’ and 3’ ends of linear RNA in close proximity for ligation and the use of ribozymatic methods in conjunction with self-splicing introns to covalently link the 5’ and 3’ ends of an in vitro-transcribed RNA molecule. However, these methods suffer from low efficiency or result in cytotoxic byproducts that must be removed using high-performance liquid chromatography (HPLC). We show that circRNA can be efficiently synthesized without cytotoxic byproducts using T4 RNA Ligase 1 and rationally designed short RNA sequences that form secondary structures designed to enhance ligation efficiency. Specifically, we show that RNA sequences that form a hairpin loop in close proximity to the 5’ and 3’ ends better support intramolecular ligation than RNA sequences without such a hairpin loop. We demonstrate that this method of ligation is applicable to a variety of RNA sequences, including long RNAs. CircRNA containing an internal ribosome entry site (IRES) 5’ to a sequence encoding green fluorescent protein is readily translated in primary human fibroblasts with minimal cytotoxicity. In summary, we present a simple, cost effective method of producing protein-encoding circRNA sequences that removes the need for complex purification methods, which may find use in biomedical applications that can benefit from extended RNA stability and translation.

To produce a targeted knock-in, a gene-editing endonuclease is used to create a double strand break (DSB) at a target site in the genome, and a plasmid donor containing a transgene as well as homology arms is inserted at the target site. However, rates of on-target integration using plasmid donors are very low, especially in primary cells and induced pluripotent stem cells (iPSCs). We hypothesized that the use of end-modified linear DNA donors could result in higher insertion rates and increased target specificity when compared with traditional plasmid donors. We synthesized end-modified linear donors using PCR with standard primers, 5’-biotinylated primers, and primers containing a 5′ polyethylene glycol (PEG) linker connected to a random 21-nucleotide single-stranded DNA sequence. We hypothesized that these donors could exhibit lower cytotoxicity, increased persistence in cells, and improved rates of on-target integration. To examine these characteristics, we compared the three donors in knock-in experiments using primary human fibroblasts and iPSCs. End-modified linear donors encoding green fluorescent protein (GFP) and a puromycin resistance gene were synthesized and electroporated into fibroblasts and iPSCs together with mRNA encoding NoveSlice gene-editing proteins targeting a sequence within the AAVS1 locus. We optimized the electroporation parameters and cell culture conditions for simultaneous delivery of gene-editing mRNA and donor DNA to iPSCs. Using GFP expression levels as a marker, the standard PCR donor resulted in the highest insertion rate, followed by the 5′ ssPEG donor and the biotinylated donor. All three donors resulted in higher integration rates than a plasmid containing the same sequence. Transfected iPSCs formed colonies of cells with uniform GFP expression that were isolated and propagated as stable knock-in lines. We show that end-modified linear donors integrate at higher rates than plasmid donors. Use of these donors may therefore represent a preferred approach for the generation of knock-in iPS cell lines.

Gene editing proteins offer an efficient means of knocking out, inserting and repairing nucleic-acid sequences in living cells. However, while gene-editing proteins can efficiently target pre-determined sequences, they can also cleave similar sequences throughout the genome, albeit with lower efficiency (so-called “off-target” editing). Many gene editing technologies include design constraints that limit the sequences that can be targeted, for example CRISPR-Cas9 requires a PAM sequence and TALENs require a thymine (T) in the zero position of the target site (“T₀”). A recently described temperature-sensitive gene-editing protein with flexible linkers, NoveSlice, shares the T₀ requirement, limiting the available target sites in regions of low sequence complexity. We sought to remove the T₀ requirement of TALEN and NoveSlice through amino-acid substitution of key residues in the N-terminal region of the DNA-binding domain. We designed and synthesized constructs encoding NoveSlice and TALEN proteins comprising various N-terminal regions using site directed mutagenesis. We tested the gene editing efficiency of these novel proteins by mRNA transfection into primary human keratinocytes. After 48 hours we amplified the target site, a region near the COL7A1 exon 73 splice acceptor site, and assessed editing using T7 endonuclease I. From this initial screen, we identified the most promising N-terminal region that had the highest editing efficiency in a target site with an N₀. Surprisingly, when measuring cutting of an N₀-containing target site, we observed “off-target” editing by TALENs while there was no editing by NoveSlice under these same conditions. Thus, the NoveSlice gene-editing protein showed a higher degree of specificity when compared to TALENs for this clinically relevant target site. These data suggest that the NoveSlice gene-editing protein can yield higher specificity than TALENs targeting the same site in primary human cells, and thus could offer an advantage in the development of both ex-vivo and in-vivo gene editing therapies.

Autologous engineered cell therapies such as autologous chimeric antigen receptor T-cell (CAR-T) therapies have revolutionized the treatment of hematologic cancers, however they are limited by manufacturing time and variability, the requirement for lymphodepletion, and side effects related to cytokine release. Allogeneic cell therapies derived from gene-edited induced pluripotent stem cells (iPSCs) are being developed to address the challenges associated with autologous engineered cell therapies. These “off-the-shelf” cell therapies contain specific edits designed to reduce immune rejection and to confer enhanced therapeutic properties and greater safety. However, efficient, footprint-free, biallelic targeting of defined loci in iPSCs remains technically challenging with current gene-editing approaches. We demonstrate efficient targeting of defined loci in iPSCs using novel messenger RNA (mRNA)-encoded gene-editing endonucleases comprising DNA-binding domains containing novel linker regions. We targeted exon 3 of beta-2 microglobulin (B2M), a key component of MHC class I molecules, and confirmed targeted editing in 10/12 lines, with 6/12 lines containing a desired biallelic deletion. Gene knockout in iPSCs was confirmed via RT-PCR and immunofluorescence in the context of B2M upregulation following exposure to interferon-γ. We show differentiation of B2M^-/- iPSCs into CD34⁺ hematopoietic progenitor cells using both 2D and 3D directed-differentiation protocols. This mRNA gene editing platform could serve an important tool for the development of minimally-immunogenic cell lines for future allogeneic cell therapies.