Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit.

Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity.

Complete motif analysis of sequence requirements for translation initiation at non-AUG start codons.

The initiation of mRNA translation from start codons other than AUG was previously believed to be rare and of relatively low impact. More recently, evidence has suggested that as much as half of all translation initiation utilizes non-AUG start codons, codons that deviate from AUG by a single base. Furthermore, non-AUG start codons have been shown to be involved in regulation of expression and disease etiology. Yet the ability to gauge expression based on the sequence of a translation initiation site (start codon and its flanking bases) has been limited. Here we have performed a comprehensive analysis of translation initiation sites that utilize non-AUG start codons. By combining genetic-reporter, cell-sorting, and high-throughput sequencing technologies, we have analyzed the expression associated with all possible variants of the -4 to +4 positions of non-AUG translation initiation site motifs. This complete motif analysis revealed that 1) with the right sequence context, certain non-AUG start codons can generate expression comparable to that of AUG start codons, 2) sequence context affects each non-AUG start codon differently, and 3) initiation at non-AUG start codons is highly sensitive to changes in the flanking sequences. Complete motif analysis has the potential to be a key tool for experimental and diagnostic genomics.

Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states.

Phenotypic heterogeneity within a population of genetically identical cells is emerging as a common theme in multiple biological systems, including human cell biology and cancer. Using live-cell imaging, flow cytometry, and kinetic modeling, we showed that two states--quiescence and cell cycling--can coexist within an isogenic population of human cells and resulted from low basal expression levels of p21, a Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21-dependent heterogeneity in cell cycle activity to double-negative feedback regulation involving CDK2, p21, and E3 ubiquitin ligases. In support of this mechanism, analysis of cells at a point before cell cycle entry (i.e., before the G1/S transition) revealed a p21-CDK2 axis that determines quiescent and cycling cell states. Our findings suggest a mechanistic role for p21 in generating heterogeneity in both normal tissues and tumors.

Tuning gene expression with synthetic upstream open reading frames.

We engineered short ORFs and used them to control the expression level of recombinant proteins. These short ORFs, encoding a two-amino acid peptide, were placed upstream of an ORF encoding a protein of interest. Insertion of these upstream ORFs (uORFs) resulted in suppression of protein expression. By varying the base sequence preceding the uORF, we sought to vary the translation initiation rate of the uORF and subsequently control the degree of this suppression. Using this strategy, we generated a library of RNA sequence elements that can specify protein expression over a broad range of levels. By also using multiple uORFs in series and non-AUG start codons, we were able to generate particularly low expression levels, allowing us to achieve expression levels spanning three orders of magnitude. Modeling supported a mechanism where uORFs shunt the flow of ribosomes away from the downstream protein-coding ORF. With a lower translation initiation rate at the uORF, more ribosomes "leak" past the uORF; consequently, more ribosomes are able to reach and translate the downstream ORF. We report expression control by engineering uORFs and translation initiation to be robust, predictable, and reproducible across all cell types tested. We propose control of translation initiation as a primary method of choice for tuning expression in mammalian systems.

Quantitative analysis of mammalian translation initiation sites by FACS-seq.

An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the -6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.

A genetic reporter system to gauge cell proliferation rate.

In higher eukaryotes, E2F transcription factors often drive expression of genes necessary for the cell cycle, notably the G1/S phase transition. With conventional transcriptional reporter systems, expression of a reporter gene from an E2F-responsive promoter would allow one to identify the fraction of cells making this transition. Here, we have engineered an E2F-responsive genetic reporter system that outputs the proliferation rate. The system takes advantage of the long half-lives of fluorescent protein reporters and output signal normalization. By doing so, it converts dynamic pulses of E2F activity into an analog output proportional to the proliferation rate. Such a system should be useful for applications involving high-throughput drug or genetic screens, investigation of cellular environment, and biological engineering.

Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers.

We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs. By specifying the sequences of the linkers, desired components can be assembled in any combination and order to generate different plasmid vectors.

AID mutates a non-immunoglobulin transgene independent of chromosomal position.

It is unknown how activation-induced cytidine deaminase (AID) targets immunoglobulin (Ig) genes during somatic hypermutation. Results to date are difficult to interpret: while some results argue that Ig genes have special sequences that mobilize AID, other work shows that non-Ig transgenes mutate. In this report, we have examined the effects of the intronic mu enhancer on the somatic hypermutation rates of a retroviral vector. For this analysis, we used centroblast-like Ramos cells to capture as much of the natural process as possible, used AIDhi and AIDlow Ramos variants to ensure that mutations are AID induced, and measured mutation of a GFP-provirus to achieve greater sensitivity. We found that mutation rates of the non-Ig provirus were AID-dependent, were similar at different genomic loci, but were approximately 10-fold lower than the V-region suggesting that AID can mutate non-Ig genes at low rates. However, the intronic mu enhancer did not increase the mutation rates of the provirus. Interestingly, exogenous over-expression of AID revealed that the V-region mutation rate can be saturated by lower levels of AID than the provirus, suggesting that selective mutation of Ig sequences is compromised in cells that over-express AID.

A murine leukemia virus with Cre-LoxP excisible coding sequences allowing superinfection, transgene delivery, and generation of host genomic deletions.

BACKGROUND: To generate a replication-competent retrovirus that could be conditionally inactivated, we flanked the viral genes of the Akv murine leukemia virus with LoxP sites. This provirus can delete its envelope gene by LoxP/Cre mediated recombination and thereby allow superinfection of Cre recombinase expressing cells. RESULTS: In our studies, the virus repeatedly infected the cell and delivered multiple copies of the viral genome to the host genome; the superinfected cells expressed a viral transgene on average twenty times more than non-superinfected cells. The insertion of multiple LoxP sites into the cellular genome also led to genomic deletions, as demonstrated by comparative genome hybridization. CONCLUSION: We envision that this technology may be particularly valuable for delivering transgenes and/or causing deletions.

Directed molecular evolution by somatic hypermutation.

After rearrangement of immunoglobulin gene segments, the immune system evolves the antibody repertoire by mutating the immunoglobulin variable region at a high rate. While this somatic hypermutation was thought to occur only at the variable region, recent studies suggest that hypermutation can occur at locations throughout the genome. Building upon this notion, we sought to exploit this mechanism as a mutagenesis tool. We created a substrate based on GFP that could be screened using flow cytometry and showed that retroviral infection can deliver the transgene to genomic locations that support hypermutation. Infected cells generated various GFP mutants with increased fluorescence intensity and analysis revealed mutations not only at the chromophore, but also an unexpected mutation at a distant residue. Our results demonstrate in principle that immunoglobulin somatic hypermutation can be a potent means of mutagenesis. With appropriate selection conditions it may be utilized to evolve gene products with desired properties.

A deep-sea hydrothermal vent isolate, Pseudomonas aeruginosa CW961, requires thiosulfate for Cd tolerance and precipitation.

Pseudomonas aeruginosa CW961, an isolate from the vicinity of a deep-sea hydrothermal vent, grew in the presence of 5 mM Cd(2+) and removed Cd(2+) from solution. Sulfate was sufficient for growth when Cd(2+) was not present in the culture medium; however, thiosulfate was necessary for Cd(2+) precipitation and cell survival in the presence of Cd(2+).

Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53.

The CRISPR (clustered regularly interspaced short palindromic repeats) platform has been developed as a general method to direct proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves and potentially mutates target genes, researchers have recently employed catalytically inactive CRISPR-associated 9 nuclease (dCas9) in order to target and repress genes without DNA cleavage or mutagenesis. With the intent of improving repression efficiency in mammalian cells, researchers have also fused dCas9 with a KRAB repressor domain. Here, we evaluated different genomic sgRNA targeting sites for repression of TP53. The sites spanned a 200-kb distance, which included the promoter, transcript sequence, and regions flanking the endogenous human TP53 gene. We showed that repression up to 86% can be achieved with dCas9 alone (i.e., without use of the KRAB domain) by targeting the complex to sites near the TP53 transcriptional start site. This work demonstrates that efficient transcriptional repression of endogenous human genes can be achieved by the targeted delivery of dCas9. Yet, the efficiency of repression strongly depends on the choice of the sgRNA target site.

Engineering ribosomal leaky scanning and upstream open reading frames for precise control of protein translation.

We have employed upstream open reading frames (uORFs) to systematically tune the translation levels of recombinant proteins. We present the design principles that guided the development of this technology and provide information that may help others in implementing synthetic uORFs for their own applications. We also report on recent applications to our own research projects, including the coupling of uORF and translation initiation site (TIS) engineering with small molecule-inducible post-translational control. Finally, we discuss opportunities to investigate and potentially engineer gene-specific translational responses to cellular stress.

Optimization of oncogene expression through intra-population competition.

Although functional roles have been assigned to many genes, e.g. those involved in cell-cycle regulation, growth signaling, or cancer, considerably less is known about the quantitative relationship between gene expression levels and outcome. We devised an intra-population competition to study oncogene dosage. Cell populations were engineered to express a range of H-Ras oncogene levels. Cells with different levels of H-Ras then "competed" for an increased share of the total cell population. Using flow cytometry to track the population composition over time, we determined the relationship between the different H-Ras oncogene expression levels and the net proliferation rate. Under culture conditions in which wild-type Ras activation was suppressed, we found that increased and maximal net proliferation occurred when the H-Ras G12V oncogene was expressed at a level 1.2-fold that of wild-type Ras. As the H-Ras G12V expression levels increased above this optimal level, proliferation rates decreased. Our findings suggest that the tumor evolution process may optimize gene expression levels for maximal cell proliferation. In principle, engineered intra-population competitions can be used to determine proliferation rates associated with the level of any ectopically expressed gene. The approach also may be used to determine proliferation rates associated with different cell species in a heterogeneous population or to improve the proliferation rate of a cell line. We also envision that the tracking of intra-population competitions could be utilized to investigate the evolution of tumors in the body.

Tetracycline-regulated expression implemented through transcriptional activation combined with proximal and distal repression.

Tetracycline-regulated expression systems are widely used to control ectopic gene expression in mammalian cells. However, background or "leaky" expression in the "off" state can limit applications that require control of expression at low levels. In this work we have engineered a tetracycline-regulated expression system with an improved range of control and lower background expression. To lower background expression without diminishing the controllable expression range, we designed a feed-forward scheme that repressed both expression of the gene of interest and the transcriptional activator. By using a tetracycline-responsive repressor that can modify chromatin and repress transcription over short and long distances, we were able to repress these two expression targets using a single tetracycline-responsive genetic element. This dual-targeting repressor/activation system demonstrated decreased background expression in its "off" state and a 25-fold range of expression in response to doxycycline. This study demonstrates that genetic circuits can be improved by leveraging trans-acting factors with long-range capabilities.

Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters.

We sought to characterize and compare wild-type and oncogenic Ras over-expression. Because different levels of Ras over-expression can have different effects on cell phenotype, it was important to evaluate a wide range of expression. Different expression levels were achieved by using retroviral vectors equipped with different strength promoters. Cells were "shotgun" transduced with a mixture of these vectors to generate heterogeneous populations exhibiting a range of expression levels. We used flow cytometry to analyze the populations and generate high-resolution, nearly continuous Ras dose-response curves. These efforts revealed that a single-copy level of oncogenic Ras generated maximal imatinib resistance and activated MAPK pathway signaling as effectively as six-fold amplification of wild-type Ras. Although further increased expression lead to even greater signal transduction, this increased expression had minimal or decreasing effects on the proliferation rate. In addition, this study introduces a general method to quantify genetic dose-response relationships and identify gene expression ranges that produce an optimized phenotypic response.

Flow cytometry of v-Abl transformed pre-B cells heterogeneous in ectopic expression levels reveals Ras dose-response.

Flow cytometry is a powerful tool for quantitative biology because it can perform single-cell analysis of large cell populations using multiple parameters. Results are often visualized as a two-dimensional scatter or contour plot. Because these plots can be relatively diffuse, it is not always straightforward to discern a relationship between measured parameters. We have demonstrated that quantitative trends can be fit to the single-cell data generated from a heterogeneous population. We engineered Abelson virus-transformed pre-B cells to express a broad range of oncogenic Ras levels. Instead of individual cultures with individual expression levels, a continuous range of levels was expressed by different cells in one heterogeneous culture. We then stained cells for downstream Erk phosphorylation to monitor MAPK signaling or employed an E2F-responsive genetic reporter to monitor cell-cycle activity. Subsequent analysis by flow cytometry and locally weighted scatterplot smoothing (LOWESS) revealed that increasing Ras oncogene expression led to increasing MAPK signaling. In contrast, E2F activity peaked at an optimal, intermediate level of Ras. To make this analytical method widely available to others, we have provided a software application that performs LOWESS on any two-parameter population data collected by flow cytometry.

Toward a bioengineered heparin: challenges and strategies for metabolic engineering of mammalian cells.

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Since Chinese hamster ovary (CHO) cells are capable of producing heparan sulfate (HS), a related polysaccharide naturally, and heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. We developed stable human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) expressing cell lines based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells. Both activity assay and disaccharide analysis showed that engineered HS attained heparin-like characteristics but not identical to pharmaceutical heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.