Publications

Mesenchymal stem cells (MSCs) are a promising cell-therapy platform with the potential to treat a diverse array of diseases due to their immunomodulator properties - properties which can be enhanced...

Mesenchymal stem cells (MSCs) are a promising cell-therapy platform with the potential to treat a diverse array of diseases due to their immunomodulator properties - properties which can be enhanced through gene editing. Gene editing autologous or donor-derived MSCs is challenging due to the non-clonal nature of these cell sources, and associated risks of off-target effects. In contrast, iPS cells, which are clonal and highly expandable, provide an ideal source of cells for gene editing and subsequent differentiation into tissue-specific cells for cell therapy applications. Here we establish stable clonal human induced pluripotent stem (iPS) cell lines engineered to express green fluorescent protein (GFP) using two different promoters. We then differentiate the engineered iPS cells to MSCs (EiMSCs) while monitoring changes in GFP expression, isolate an EiMSC subpopulation and verify differentiation using surface markers. Single-stranded DNA (ssDNA)donors encoding GEP under the control of the Jel’ or EFla promoters were inserted into iPS cells using mRNA encoding UltraSlice gene-editing proteins targeting the AAVS] safe-harbor locus. GFP transgene insertion rates of 40% and 10% were observed for Jel" and EFlapromoter-containing donors, respectively. While EFla and JeT promoters drive robust GFP expression when inserted directly in iMSCs, in iP$ cells strong GFP expression was only observed under EFla - expression was not detected in Je'l-GEP iPS cells. Clonal cell lines were generated from both lines using single-cell deposition, and bi-allelic insertion into the AAVS1 locus was verified by amplicon sequencing. Engineered iPS cells were differentiated into EiMSCs, and GEP expression was monitored. During differentiation, the number of GFP-expressing cells decreased from >99% in the starting EF1a-GEP iPS cells to 40% in the differentiated EFla-GEP iMSCs. In contrast, J¢'I-GFP iPS$ cells began expressing GFP during differentiation but stopped expressing GFP near the end of the differentiation process. Treatment with 'I'richostatin A, a selective histone deacetylase (HDAC) inhibitor, resulted in a temporary increase in GFP expression in JeT-GFP cells during differentiation. To obtain a clonal population of EiMSCs that uniformly express GFP, we then enriched the GFP-expressing EF1a-GEP iMSCs, resulting in a cell line that exhibited traditional MSC surface markers (positive markers: CD90, CD73, CD105, and CD44; negativemarkers:CD34, TRA-1-60, 1'RA-1-81, CD45, and HLA-DR) and displayed stable GFP expression for over 7 passages. Here we demonstrate a platform for developing clonal EiMSC cell populations that uniformly and stably express a desired protein from a transgene inserted into a defined genomic locus by mRNA gene editing. We also show temporal control of transgene expression using small molecules during directed differentiation of iPS cells. This platform benefits from high knock-in efficiency enabled by mRNA gene editing combined with ssDNA donors and may prove useful for the development of cell therapies engineered to express therapeutic proteins.

In recent years, lipid-based mRNA delivery has become the gold standard method for inducing exogenous protein production in vivo as evidenced by the success of the COVID-19 mRNA vaccines. While...

In recent years, lipid-based mRNA delivery has become the gold standard method for inducing exogenous protein production in vivo as evidenced by the success of the COVID-19 mRNA vaccines. While intramuscular injections are ideal for vaccine applications, intravenous injections are generally more suitable for achieving broad internal distribution of therapeutic payloads. Following systemic administration, blood cells are the first cells encountered by lipid nanoparticles (LNPs) and therefore serve as high-interest targets for in situ protein production. With the goal of identifying a lipid formulation capable of efficiently transfecting blood cells, we synthesized and screened a library of 16 multivalent ionizable lipids with variations in headgroup and lipid tail. Headgroup variations included spermine (a naturally occurring biomolecule), dihydroxyspermine, 2- hydroxypropylamine, and ethanolamine (the headgroup of SM-102, the ionizable lipid component of Spikevax). The lipid tails varied in degree of unsaturation, carbon chain length, and head-to-tail spacer length. Lipids were characterized as lipoplexes and as solid LNPs, using mRNA encoding the green fluorescent protein (GFP) as representative nucleic acid cargo. Nanoparticle size, surface charge, mRNA encapsulation, transfection efficiency, and cellular toxicity were evaluated. Lipoplexes were formulated at various weight ratios of lipid/mRNA, and mRNA-loading efficiency was determined via gel electrophoresis. The lowest lipid/mRNA weight ratio that displayed complete complexation for each lipid was used for the corresponding lipids in subsequent in vitro analyses. Notably, the greatest transfection efficiency in the lipoplex form was induced by FB3-54, a lipid comprised of a spermic headgroup and bis hexyl 2-hexyldecanoate tails. FB3-54 lipoplexes enabled efficient transfection of GFP mRNA in the THP-1 human monocyte cell line and exhibited a half-maximal effective concentration (EC50) of 147 ng RNA per 50,000 cells, which was lower than that of Lipofectamine 3000 (242 ng RNA per 50,000 cells). The measured diameter of the FB3- 54 lipoplexes was 359 nm. FB3-54 also functioned effectively as a solid LNP when using a molar ratio of 50:38.5:10:1.5 (ionizable lipid/cholesterol/ DSPC/DMG-PEG2000) and a 0.12 weight ratio of mRNA to ionizable lipid. FB3-54 LNPs were 170 nm in diameter, possessed a surface charge of +3.6 mV (at a pH of 7.0), demonstrated near complete encapsulation efficiency, and exhibited greater in vitro transfection efficiency than the clinically used Dlin-MC3-DMA LNPs. The ability to efficiently target blood cells using lipid delivery systems opens the door to a variety of applications, including potent in vivo mRNA delivery and cell reprogramming,

Induced pluripotent stem (iPS) cell therapies have the potential to treat a wide variety of devastating diseases. iPS cell-derived lymphocytes (c.g., T cells and NK cells) engineered to express targeting...

Induced pluripotent stem (iPS) cell therapies have the potential to treat a wide variety of devastating diseases. iPS cell-derived lymphocytes (c.g., T cells and NK cells) engineered to express targeting molecules such as chimeric antigen receptors (CARs) have shown clinical promise to treat hematological malignancies. More recently, iPS cell-derived myeloid cells are being developed to treat both hematological malignancies and solid tumors due to the ability of these cells to infiltrate and modulate the tumor microenvironment. Despite preliminary success, several challenges still remain, including poor infiltration of cytotoxic lymphocytes into solid tumors and insufficient cytotoxicity of myeloid cells. We hypothesized that a multi-cell-type therapy comprising both lymphocyte and myeloid cells may work synergistically, enhancing cytotoxicity and efficacy. We previously demonstrated the directed differentiation of mRNA reprogrammed iPS cells into functional cytotoxic lymphocytes and monocyte-derived M1 and M2 polarized macrophages. Here we describe a scalable bioreactor-based process for parallel differentiation of mRNA reprogrammed iPS cells into both CD14+ (>95% positive) macrophages and CD56bright/CD16dim NK cells. "This process yields 1x10"6 myeloid cells/ml and 3x10"5 lymphoid cells/ml, and is amenable to scaling to clinically relevant doses. In vitro, the lymphoid and myeloid cells showed synergistic tumor cell killing of SKOV3 ovarian cancer cells (combined: 15.6%; macrophage alone = 2.2% (p<0.01); NK alone = 7.5% (p<0.05); EI = 5:1). The combined cells showed increased expression of TNF-a, and demonstrated enhanced clustering and tumor cell engagement. To further improve the macrophages’ ability to target and infiltrate solid tumors, we transfected macrophages with mRNA encoding a humanized ROR1-CAR protein. mRNA transfection increased cytotoxicity towards SKOV3 cells by 6-fold. Here we describe a scalable platform for generating iPS cell-derived multi-cell-type therapies comprising both lymphoid and myeloid cells. We demonstrate that, much like the natural cellular immune response, these cells act synergistically to kill tumor cells in vitro. By more closely mimicking natural cellular immunity, multi-cell-type cell therapies represent a new class of cell therapies that may play an important role in the development of new medicines to treat cancer.

Lipid nanoparticles (LNPs) containing cationic or ionizable lipids offer several advantages compared to other vehicles for nucleic acid delivery and have seen expanded clinical use with the introduction the COVID-19...

Lipid nanoparticles (LNPs) containing cationic or ionizable lipids offer several advantages compared to other vehicles for nucleic acid delivery and have seen expanded clinical use with the introduction the COVID-19 mRNA vaccines and in treatments for genetic diseases. However, poor targeting, insufficient cellular uptake, and low endosomal release currently limit the use of lipid nanoparticles as efficient delivery systems for next-generation gene therapies. 'L'o address these limitations, we designed, synthesized, and formulated novel ionizable lipids as LNPs to optimize mRNA delivery to cells. We developed a library of 12 ionizable lipids containing hexyl 2-hexyldecanoate lipid tails and hydrophilic headgroups derived from spermine or 2- hydroxypropylamine. Nanoparticle formulations incorporating these ionizable lipids were prepared at a molar ratio of 50:38.5:10:1.5 (ionizable lipid: cholesterol: DSPC/DOPE: DMG-PEG2000) with mRNA encoding GFP at an N/P ratio of 3. For all the lipids tested, a mean diameter ranging from 100-150nm was measured by DLS. All of the lipids showed encapsulation efficiencies between 70-80%, comparable to LNPs formulated using FDA-approved lipids ALC0315 and DLin-MC3-DMA. LNPs were administered to iPSC-derived MSCs, primary human fibroblasts, and keratinocytes. Microplate imaging showed peak GFP fluorescence between 40- and 48-hours post-transfection. We identified novel ionizable lipids that produced LNPs exhibiting lower mean particle sizes, higher loading efficiencies, and enhanced GEP expression compared to LNPs incorporating ALC0315 or DLin-MC3-DMA. These results indicate that lipids containing elements of spermine and 2-hydroxypropylamine headgroups may serve as components of next-generation, lipid nanoparticle-based gene therapies and assist in the rational design of ionizable lipids for mRNA delivery.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an inducible, heme-containing enzyme that is critically involved in tryptophan catabolism and known to be a prominent immune regulator. Cell therapies with increased IDO1 expression...

Indoleamine 2,3-dioxygenase 1 (IDO1) is an inducible, heme-containing enzyme that is critically involved in tryptophan catabolism and known to be a prominent immune regulator. Cell therapies with increased IDO1 expression are of high interest for a variety of indications, including autoimmune disorders, inflammatory diseases, transplant recovery, and wound healing. In particular, iPSC-derived mesenchymal stem cells (iIMSCs) engineered to overexpress 1DO1 may be ideal for suppressing dysregulated immune cells while simultaneously promoting expansion of regulatory I' lymphocytes and the M1 (pro- inflammatory) to M2 (anti-inflammatory) polarization of macrophages. Here, we report the development of an iMSC cell line containing an IDOI transgene under the control of a Jel’ promoter that was inserted into the AAVS] safe-harbor locus in mRNA-reprogrammed iPSCs. A clonal population of edited cells (i.e. IDO1-1PSCs) was isolated using single-cell sorting. Bi-allelic insertion of the IDOI transgene was confirmed by amplicon sequencing. The 1DO1-1PSCs were then differentiated to IDO1-iMSCs. During differentiation, we found that the 1DO1-iPSCs showed an unexpected, cuboidal morphology and noticeably decreased proliferation rates relative to control iPSCs, possibly indicating that IDO1 was interfering with the differentiation process. 1'wo small-molecule IDO] inhibitors, Epacadostat, which binds to holo (heme-bound) IDOI and inhibits the enzymatic function of the protein, and IDO1-IN-5, which binds to apo (heme-dissociated) 1DO1 and inhibits [DO1-dependent cell signaling, were added to the culture for the final five days of differentiation. The addition of both IDO! inhibitors (each at a 10uM concentration) increased the proliferation of the partly differentiated IDO1-iPSCs (6 h reduction in doubling time) with a population doubling time approaching that of control iMSCs (29 h vs. 27 h). While both inhibitors could enhance proliferation independently, Epacadostat alone had a larger effect on population doubling time than the IDO1-IN-5 alone (6h vs. 2h reduction in doubling time), suggesting that the anti-inflammatory enzymatic function of IDO1 may be most responsible for decreased proliferation during differentiation of IDO1-iPSCs. Notably, we also found that when administering both IDO1 inhibitors to non-engineered control iMSCs, the population doubling time increased relative to untreated test cultures (34 h versus 27 h), suggesting a possible minimum IDO1 protein level required for optimal iMSC culture. These results suggest that small-molecule inhibition of transgene-encoded proteins may form a key element of directed differentiation process development for knock-in iP$ cell lines, and may be useful for the scale-up manufacturing of IDO1-iMSCs in particular.

Macrophages’ ability to infiltrate solid tumors and engage in both direct killing of cancer cells and recruitment of other immune cells has made them a promising target for development of...

Macrophages’ ability to infiltrate solid tumors and engage in both direct killing of cancer cells and recruitment of other immune cells has made them a promising target for development of next-generation cancer immunotherapies. The innate ability of macrophages to ingest foreign genetic material also facilitates their engineering with formulated nucleic acids, including mRNA. ‘The oncoantigen tyrosine-protein kinase transmembrane receptor ROR1 has garnered interest for its minimal expression in healthy adult cells and overexpression in many malignancies, including solid tumors associated with ovarian, lung, and triple-negative breast cancer. Here, we present an mRNA-based platform for rapid prototyping of macrophage engineering approaches. We show mRNA delivery to peripheral blood mononuclear cell (PBMC) and iPS cell-derived macrophages for gene editing prototyping and functional assessment of encoded proteins. To develop this platform, we transfected macrophages with unmodified or 5-methoxyuridine (5-moU)-containing mRNA encoding green fluorescent protein (GFP). Both mRNAs resulted in more than 95% of cells displaying GFP within 4 hours. We next designed an ROR1-targeting CAR with a CD3 zeta activation domain and 4-1BB costimulatory domain. Transfection of mRNA encoding the ROR1-CAR yielded 70% CAR-expressing cells, as measured using PE-labelled ROR1. We then compared ROR] affinity of rabbit and humanized binding domains and found that the humanized binding domain displayed a 2.5-fold increase in affinity as measured by flow cytometry using PE-labelled RORI1. ‘The human receptor domain, but not the rabbit domain, demonstrated activation when bound to ROR1 as assessed by immunofluorescence of CD3 zeta phosphorylation. We also assessed the mRNA-encoded ROR1-CAR's functionality by measuring killing of ROR1-expressing SKOV-3 ovarian cancer cells. Both the rabbit and humanized ROR1 domains of the CAR displayed significantly increased cytotoxicity towards SKOV-3 cells when compared with untransfected macrophages after a 24-hour co-culture at a 5:1 effector-to-target ratio (p<0.01). We then inserted the ROR1-CAR sequence into the AAVSI safe harbor locus of iPS cells under the control of an SFC promotor, isolated5 biallelic-inserted lines, and differentiated them into macrophages. These results demonstrate an mRNA-based platform for rapid prototyping of macrophage engineering approaches. I'ransfection of macrophages with mRNA encoding a chimeric antigen receptor (CAR) targeting ROR1 resulted in functional expression in vitro, facilitating optimization of the antibody and co-stimulatory domains to improve protein binding affinity and immune activation. This platform thus enables the assessment and validation of novel macrophage gene editing strategies and is being explored for the development of macrophage-engineering therapies for solid tumor applications.

Many gene editing strategies involve the use of nucleases to generate targeted double-strand breaks (DSBs) in genomic DNA, which is often associated with cytotoxicity and off-target effects that can prevent...

Many gene editing strategies involve the use of nucleases to generate targeted double-strand breaks (DSBs) in genomic DNA, which is often associated with cytotoxicity and off-target effects that can prevent clinical translation. Such undesirable outcomes have led to the development of gene-editing nickases, which instead create targeted single-strand breaks (SSBs) that favor high-fidelity repair through the homology-directed repair (HDR) pathway rather than the more error-prone non-homologous end joining (NHEJ) pathway. Here, we explore the use of UltraSlice gene editing proteins containing cleavage domain variants with nickase functionality for targeted insertion of donor sequences into a defined genomic locus. Using UltraSlice gene editing proteins targeting exon 73 in COL7AI (mutations in which cause dystrophic epidermolysis bullosa), we tested combinations of 3 mutations previously reported to confer nickase functionality to the catalytic domain of Fokl, a I'ype IS restriction endonuclease (D4504A, D450N, and 1D467A). Notably, we found that D450A and D450N resulted in significantly reduced NHE] relative to the COL7A1_e73 UltraSlice pair with a wild-type Fokl cleavage domain, with D450N exhibiting the least amount of NHE). We confirmed these results with Sanger Sequencing, comparing the D450N-treated PCR amplicon to a wild-type COL7AI PCR amplicon and observed no significant alteration of the genomic target site. We then compared the ability of nickases to insert a 300 bp dsDNA donor sequence via electroporation into primary human fibroblasts. Insertion band intensities showed high insertion efficiencies for D450A and D450N (58.7% and 42.9%, respectively), though lower than standard UltraSlice (72.3%). Our data demonstrate that gene-editing nickases enable scarless insertion of donor sequences into defined genomic loci, and thus may have the potential to improve the safety of in vivo gene insertion by reducing off-target effects.

Genome-editing technology provides a means of modifying genes in living cells, and is being explored for the development of therapies to treat cancer and a variety of genetic disorders. Gene-editing...

Genome-editing technology provides a means of modifying genes in living cells, and is being explored for the development of therapies to treat cancer and a variety of genetic disorders. Gene-editing proteins can be used to create single- and double-strand breaks at specific genomic sites for knocking out a gene or, when combined with an exogenous DNA donor, knock-in of a defined sequence. Compared with double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) exhibits lower toxicity and is less prone to random genomic integration, making it an attractive form of DNA donor for gene knock-in. However, current methods of synthesizing ssDNA, including enzymatic digestion, asymmetric PCR, and chemical synthesis, suffer from low yields, contamination with residual dsDNA, length limitations, and high cost. Here, we present an enzymatic approach for producing long (>3kb) and concentrated (>1ug/uL) ssDNA suitable for generation of knock-in lines of human induced pluripotent stem (hiPS) cells. We PCR-amplified dsDNA from plasmid templates using 5-modified primers incorporating a 5’ phosphate on the strand intended for digestion and a single 5’phosphorothioate linkage on the protcin-cncoding strand to protect against digestion. The resulting PCR products were treated with lambda exonuclease, which preferentially digests strands with a 5"phosphate group, to yield a single-stranded product. ''o minimize degradation of the ssDNA, we included in the reaction a short, double-stranded oligonucleotide (dsDecoy), for which lambda exonuclease has a higher affinity than ssDNA. Reactions were further treated with the less processive 17 exonuclease to climinate residual dsDNA not digested by lambda exonuclease, yielding concentrated (>1ug/ulL) ssDNA. Gel electrophoresis analysis of five products ranging from 1.4kbto 3.3kb revealed a single, sharp band in the region of interest. Four of the ssDNA products contained less than 0.3% residual dsDNA by mass, as determined by gel electrophoresis using a double-stranded standard of known concentration. The fifth, 3.3kb ssDNA product contained 1.1% dsDNA by mass. To test their utility as knock-in donors, ssDNA products were co-electroporated into hiPS$ cells with mRNA encoding UltraSlice gene-editing proteins targeting the AAVS1 safe harbor locus. Insertion efficiencies were 67.8% for a 1.2kb donor, 8.6% for a 2kb donor encoding a RORI CAR, and 2.7% for a 2.8kb donor encoding green fluorescent protein. In summary, we demonstrate a method for high-yield synthesis of ssDNA suitable for cellular applications, including mRNA gene editing-mediated knock-in in iPS$ cells. Overall, this platform may prove useful in the development of gene-editing therapeutics.

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...

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,...

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.