Isolation and characterization of human cells resistant to retrovirus infection

<p>Abstract</p> <p>Background</p> <p>Identification of host cell proteins required for HIV-1 infection will add to our knowledge of the life cycle of HIV-1 and in the development of therapeutics to combat viral infection. We and other investigators have mutagenized rodent cells and isolated mutant cell lines resistant to retrovirus infection. Since there are differences in the efficiency of single round infection with VSVG pseudotyped HIV-1 on cells of different species, we conducted a genetic screen to isolate human cells resistant to HIV-1 infection. We chemically mutagenized human HeLa cells and validated our ability to isolate mutants at test diploid loci. We then executed a screen to isolate HeLa cell mutants resistant to infection by an HIV-1 vector coding for a toxic gene product.</p> <p>Results</p> <p>We isolated two mutant cell lines that exhibit up to 10-fold resistance to infection by HIV-1 vectors. We have verified that the cells are resistant to infection and not defective in gene expression. We have confirmed that the resistance phenotype is not due to an entry defect. Fusion experiments between mutant and wild-type cells have established that the mutations conferring resistance in the two clones are recessive. We have also determined the nature of the block in the two mutants. One clone exhibits a block at or before reverse transcription of viral RNA and the second clone has a retarded kinetic of viral DNA synthesis and a block at nuclear import of the preintegration complex.</p> <p>Conclusion</p> <p>Human cell mutants can be isolated that are resistant to infection by HIV-1. The mutants are genetically recessive and identify two points where host cell factors can be targeted to block HIV-1 infection.</p

Systems engineering N-glycans of recombinant therapeutic proteins

Protein N-glycosylation reactions form a distributed reaction network spanning over different compartments of Golgi apparatus. The resulting glycan structures are influenced by glycosylation enzymes, the supply rate of nucleotide sugars, as well as competition among extending glycan substrates for a common enzyme and among different enzymes for a common substrate. Controlling the glycan profile of a therapeutic protein product is important for product quality for both innovative drugs as well as biosimilars. Metabolic engineering of the glycosylation pathway offers a venue for modulating the glycan profile. We have taken a systems engineering approach to identify, through model assisted design, the genetic manipulations that may steer the glycan flux to the desired path. However, unlike the energy metabolism pathway for which a small number of enzymes play pivotal roles in controlling the flux, the glycosylation pathway lacks key regulated steps as easily identifiable targets for genetic alteration to re-direct the flux. The model prediction thus serves only as a imprecise guide rather than a clear beacon. Furthermore, very likely multiple genetic alterations are needed in order to steer glycan flux distribution. A scheme of rapid construction of gene combinations to facilitate genetic engineering of the cell is necessary. We establish a golden gate assembly workflow for production of multi-gene constructs for engineering the glycan biosynthesis pathway. Libraries containing promoters of varying strengths, terminators, and glycosylation related coding sequences of interest, all refactored to be devoid of type IIS restriction sites, were synthesized. In the first level of assembly, an additional library of single gene constructs were formed from these base components with single reactions. In the second level of assembly, these monocistrons were then combinatorically combined to form a multi-gene cassette library. In an application of this approach, the N-glycosylation pattern of a recombinant IgG produced in CHO cells was examined with a stoichiometric network visualization tool (GlycoVis) to track the reaction paths which lead to the product glycans and identify galactosylation as potentially limiting glycan maturation. Cassettes consisting of sequences coding for nucleotide sugar synthesis enzymes, nucleotide sugar transporters, and glycosyltransferases were then selected to engineer the IgG producing cell. Multiple cassettes successfully directed the glycosylation to produce antibody with desirable glycoforms. These results served to refine our model parameters and sharpen its predictive capabilities. This combination of systems analysis and synthetic glycoengineering can be broadly applied and enhances our capability to steer N-Glycan patterns and control the quality of therapeutic proteins

Systems engineering of a CHO cell line for enhanced process robustness

Recent advances in genome engineering have opened great opportunities for engineering Chinese hamster ovary cells. An ideal cell line is no longer just one with high productivity, but also with high stability in both the productivity and product quality, and in both extensive passaging and long term continuous culture. Furthermore, an ideal cell line should be provided with process controllability, allowing the use of environmental control variables to steer its metabolism to a reaction pathway that favors the synthesis of product with the desired quality attributes. Importantly, these superior traits must be genetically and epigenetically stable and be passed on to new production lines in cell line development. We have taken a systems approach that integrates genomic information and metabolic model predictions to devise a strategy and to develop tools for attaining those goals. In tool development we reassembled the Chinese hamster genome and combined different versions of the genome to identify consensus segments as high confidence regions and annotated the genome. An expression microarray and a comparative genomic hybridization array for gene coding regions were designed to facilitate cell engineering studies. Using solution phase capture and nested PCR we also established methods of rapidly identifying the integration sites of transgenes on the genome. An induced pluripotent stem cell (iPSC) line was derived from Chinese hamster embryonic fibroblasts for use as control in genomic and epigenomic tool development. Furthermore, we extended our kinetic model for cell metabolism to link with the glycosylation model and now embark on devising a reduced model suitable for systems optimization. The study involves surveying an established producing line and creating cell lines with a single copy transgene of GFP reporter or of IgG-GFP. The design of the single copy line entails a swappable recombination site for exchange of transgene so that cell lines which are otherwise “identical” but with different transgenes can be systematically compared. Through meta-analysis of archived transcriptome data we identified genes with different dynamics of expression patterns that can be useful for the dynamic control of cell behavior. CRISPR/Cas9 was employed to knock in a GFP between the first exon of the TXNIP gene and its endogenous promoter. Interestingly, the transcript levels of GFP in all investigated clones fell in a small range, but the dynamic profile was variable among them. In single copy clones of GFP reporter and IgG transgene, the transcript levels varied widely reflecting the probabilistic nature of their integration in the genome. The IgG titer and their transcript level of the clones also varied over a wide range. Overall, the result does not reveal a correlation between the transgene transcript level and the expression of the gene at the locus. Importantly, a number of clones showed a very high IgG transcript level, consistent with our previous report of a high transgene level prior to transgene amplification. The genomic context that may contribute to the variability, e.g. genomic consistency among the clones in terms of chromosome number, karyotype, and CGH, is being investigated. The implications of our findings to date and our work on implementing metabolic model prediction in this designing CHO cell line will be discussed. Although this work represents only an early step toward system engineering to cell line development, we believe such approaches will open new avenues to engineer cell lines and influence process development of biologics manufacturing in the coming years

A synthetic biology based cell line engineering pipeline

An ideal host cell line for deriving cell lines of high recombinant protein production should be stable, predictable, and amenable to rapid cell engineering or other forms of phenotypical manipulation. In the past few years we have employed genomic information to identify “safe harbors” for exogenous gene integration in CHO cells, deployed systems modeling and optimization to design pathways and control strategies to modify important aspects of recombinant protein productivity, and established a synthetic biology approach to implement genetic changes, all with the goal of creating a pipeline to produce “designer” cell lines. Chinese hamster ovary (CHO) cells are the preferred platform for protein production. However, the Chinese hamster genome is unstable in its ploidy, is subject to long and short deletions, duplications, and translocations. In addition, gene expression is subject to epigenetic changes including DNA methylation, histone modification and heterochromatin invasion, thus further complicating transgene expression for protein production in cell lines. With these issues in mind, we set out to engineer a CHO cell line highly amenable to stable protein production using a synthetic biology approach. We compiled karyotyping and chromosome number data of several CHO cell lines and sublines, identified genomic regions with high a frequency of gain and loss of copy number using comparative genome hybridization (CGH), and verified structural variants using sequencing data. We further used ATAC (Assay for Transposase-Accessible Chromatin) sequencing to study chromatin accessibility and epigenetic stability within the CHO genome. RNA-seq data from multiple cell lines were also used to identify regions with high transcriptional activity. Analysis of these data allowed the identification of several “safe harbor” loci that could be used for cell engineering. Based on results of the data analysis and identification of “safe harbors”, we engineered an IgG producing cell line with a single copy of the product transgene as a template cell line. This product gene site is flanked by sequences for recombinase mediated cassette exchange, therefore allowing easy substitution of the IgG producing gene for an alternative product gene. Furthermore, a “landing pad” for multi-gene cassette insertion was integrated into the genome at an additional site. Together, these sites allowed engineering of new cell lines producing a fusion protein and Erythropoietin to be generated from the template cell line. To enable rapid assembly of product transgenes and genetic elements for engineering cell attributes into multi-gene cassettes, we adopted a golden-gate based synthetic biology approach. The assembly of genetic parts into multi-gene cassettes in a LEGO-like fashion allowed different combinations of genes under the control of various promoters to be generated quickly for introduction into the template cell line. Using this engineered CHO cell line, we set out to study metabolism and product protein glycosylation for cell engineering. To guide the selection of genetic elements for cell engineering, we developed a multi-compartment kinetic model, as well as a flux model of energy metabolism and glycosylation. The transcriptome meta-data was used extensively to identify genes and isoforms expressed in the cell line and to estimate the enzyme levels in the model. The flux model was used to identify and the LEGO-like platform was used to implement the genetic changes that can alter the glycosylation pattern of the IgG produced by the template cell line. Concurrently we employed a systems optimization approach to identify the genetic alterations in the metabolic pathway to guide cell metabolism toward a favorable state. The model prediction is being implemented experimentally using the synthetic biology approach. In conclusion, we have illustrated a pipeline of rational cell line engineering that integrates genomic science, systems engineering and synthetic biology approaches. The promise, the technical challenges and possible limitations will be discussed in this presentation

Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online

Characterization of resistance to rhabdovirus and retrovirus infection in a human myeloid cell line.

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1

Parental and mutant 30-2 and 42-7 cells were seeded and growth measured over time with the MTT assay

<p><b>Copyright information:</b></p><p>Taken from "Isolation and characterization of human cells resistant to retrovirus infection"</p><p>http://www.retrovirology.com/content/4/1/45</p><p>Retrovirology 2007;4():45-45.</p><p>Published online 3 Jul 2007</p><p>PMCID:PMC1925114.</p><p></p> . The extent of integrated HIV-1 vector was measured by infection of cells at moi = 1. The cells were passaged 3 times and the quantity of stable HIV-1 DNA was measured by quantitative real time PCR