SENTI-101, a Preparation of Mesenchymal Stromal Cells Engineered to Express IL12 and IL21, Induces Localized and Durable Antitumor Immunity in Preclinical Models of Peritoneal Solid Tumors.

Advanced peritoneal carcinomatosis including high-grade ovarian cancer has poor prognoses and a poor response rate to current checkpoint inhibitor immunotherapies; thus, there is an unmet need for effective therapeutics that would provide benefit to these patients. Here we present the preclinical development of SENTI-101, a cell preparation of bone marrow-derived mesenchymal stromal (also known as stem) cells (MSC), which are engineered to express two potent immune-modulatory cytokines, IL12 and IL21. Intraperitoneal administration of SENTI-101 results in selective tumor-homing and localized and sustained cytokine production in murine models of peritoneal cancer. SENTI-101 has extended half-life, reduced systemic distribution, and improved antitumor activity when compared with recombinant cytokines, suggesting that it is more effective and has lower risk of systemic immunotoxicities. Treatment of tumor-bearing immune-competent mice with a murine surrogate of SENTI-101 (mSENTI-101) results in a potent and localized immune response consistent with increased number and activation of antigen presenting cells, T cells and B cells, which leads to antitumor response and memory-induced long-term immunity. Consistent with this mechanism of action, co-administration of mSENTI-101 with checkpoint inhibitors leads to synergistic improvement in antitumor response. Collectively, these data warrant potential clinical development of SENTI-101 for patients with peritoneal carcinomatosis and high-grade ovarian cancer.Graphical abstract: SENTI-101 schematic and mechanism of actionSENTI-101 is a novel cell-based immunotherapeutic consisting of bone marrow-derived mesenchymal stromal cells (BM-MSC) engineered to express IL12 and IL21 intended for the treatment of peritoneal carcinomatosis including high-grade serous ovarian cancer. Upon intraperitoneal administration, SENTI-101 homes to peritoneal solid tumors and secretes IL12 and IL21 in a localized and sustained fashion. The expression of these two potent cytokines drives tumor infiltration and engagement of multiple components of the immune system: antigen-presenting cells, T cells, and B cells, resulting in durable antitumor immunity in preclinical models of cancer.

Engineering dynamical control of cell fate switching using synthetic phospho-regulons.

Many cells can sense and respond to time-varying stimuli, selectively triggering changes in cell fate only in response to inputs of a particular duration or frequency. A common motif in dynamically controlled cells is a dual-timescale regulatory network: although long-term fate decisions are ultimately controlled by a slow-timescale switch (e.g., gene expression), input signals are first processed by a fast-timescale signaling layer, which is hypothesized to filter what dynamic information is efficiently relayed downstream. Directly testing the design principles of how dual-timescale circuits control dynamic sensing, however, has been challenging, because most synthetic biology methods have focused solely on rewiring transcriptional circuits, which operate at a single slow timescale. Here, we report the development of a modular approach for flexibly engineering phosphorylation circuits using designed phospho-regulon motifs. By then linking rapid phospho-feedback with slower downstream transcription-based bistable switches, we can construct synthetic dual-timescale circuits in yeast in which the triggering dynamics and the end-state properties of the ON state can be selectively tuned. These phospho-regulon tools thus open up the possibility to engineer cells with customized dynamical control.

Modular engineering of cellular signaling proteins and networks.

Living cells respond to their environment using networks of signaling molecules that act as sensors, information processors, and actuators. These signaling systems are highly modular at both the molecular and network scales, and much evidence suggests that evolution has harnessed this modularity to rewire and generate new physiological behaviors. Conversely, we are now finding that, following nature's example, signaling modules can be recombined to form synthetic tools for monitoring, interrogating, and controlling the behavior of cells. Here we highlight recent progress in the modular design of synthetic receptors, optogenetic switches, and phospho-regulated proteins and circuits, and discuss the expanding role of combinatorial design in the engineering of cellular signaling proteins and networks.

Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors.

The Notch protein is one of the most mechanistically direct transmembrane receptors-the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus, multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.

Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase.

The development of new methods for gene addition to mammalian genomes is necessary to overcome the limitations of conventional genetic engineering strategies. Although a variety of DNA-modifying enzymes have been used to directly catalyze the integration of plasmid DNA into mammalian genomes, there is still an unmet need for enzymes that target a single specific chromosomal site. We recently engineered zinc-finger recombinase (ZFR) fusion proteins that integrate plasmid DNA into a synthetic target site in the human genome with exceptional specificity. In this study, we present a two-step method for utilizing these enzymes in any cell type at randomly-distributed target site locations. The piggyBac transposase was used to insert recombinase target sites throughout the genomes of human and mouse cell lines. The ZFR efficiently and specifically integrated a transfected plasmid into these genomic target sites and into multiple transposons within a single cell. Plasmid integration was dependent on recombinase activity and the presence of recombinase target sites. This work demonstrates the potential for broad applicability of the ZFR technology in genome engineering, synthetic biology and gene therapy.

Structure-guided reprogramming of serine recombinase DNA sequence specificity.

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences > 10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with > 80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.

Directed evolution of recombinase specificity by split gene reassembly.

The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding beta-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.

Synthesis of programmable integrases.

Accurate modification of the 3 billion-base-pair human genome requires tools with exceptional sequence specificity. Here, we describe a general strategy for the design of enzymes that target a single site within the genome. We generated chimeric zinc finger recombinases with cooperative DNA-binding and catalytic specificities that integrate transgenes with >98% accuracy into the human genome. These modular recombinases can be reprogrammed: New combinations of zinc finger domains and serine recombinase catalytic domains generate novel enzymes with distinct substrate sequence specificities. Because of their accuracy and versatility, the recombinases/integrases reported in this work are suitable for a wide variety of applications in biological research, medicine, and biotechnology where accurate delivery of DNA is desired.

Evolution of programmable zinc finger-recombinases with activity in human cells.

Site-specific recombinases are important tools for genomic engineering in many living systems. Applications of recombinases are, however, constrained by the DNA targeting endemic of the recombinase used. A tremendous range of recombinase applications can be envisioned if the targeting of recombinase specificity can be made readily programmable. To address this problem we sought to generate zinc finger-recombinase fusion proteins (Rec(ZF)s) capable of site-specific function in a diversity of genetic contexts. Our first Rec(ZF), Tn3Ch15(X2), recombined substrates derived from the native Tn3 resolvase recombination site. Substrate Linked Protein Evolution (SLiPE) was used to optimize the catalytic domains of the enzymes Hin, Gin, and Tn3 for resolution between non-homologous sites. One of the evolved clones, GinL7C7, catalyzed efficient, site-specific recombination in a variety of sequence contexts. When introduced into human cells by retroviral transduction, GinL7C7 excised a 1.4 kb EGFP cassette out of the genome, diminishing fluorescence in approximately 17% of transduced cells. Following this template of rational design and directed evolution, Rec(ZF)s may eventually mediate gene therapies, facilitate the genetic manipulation of model organisms and cells, and mature into powerful new tools for molecular biology and medicine.