Application of a simple unstructured kinetic and cost of goods models to support T-cell therapy manufacture

Manufacturing of cell therapy products requires sufficient understanding of the cell culture variables and associated mechanisms for adequate control and risk analysis. The aim of this study was to apply an unstructured ordinary differential equationbased model for prediction of T-cell bioprocess outcomes as a function of process input parameters. A series of models were developed to represent the growth of T-cells as a function of time, culture volumes, cell densities, and glucose concentration using data from the Ambr®15 stirred bioreactor system. The models were sufficiently representative of the process to predict the glucose and volume provision required to maintain cell growth rate and quantitatively defined the relationship between glucose concentration, cell growth rate, and glucose utilization rate. The models demonstrated that although glucose is a limiting factor in batch supplied medium, a delivery rate of glucose at significantly less than the maximal specific consumption rate (0.05 mg 1 ? 106 cell h1 ) will adequately sustain cell growth due to a lower glucose Monod constant determining glucose consumption rate relative to the glucose Monod constant determining cell growth rate. The resultant volume and exchange requirements were used as inputs to an operational BioSolve cost model to suggest a cost-effective T-cell manufacturing process with minimum cost of goods per million cells produced and optimal volumetric productivity in a manufacturing settings. These findings highlight the potential of a simple unstructured model of T-cell growth in a stirred tank system to provide a framework for control and optimization of bioprocesses for manufacture

Engineering Buffering and Hydrolytic or Photolabile Charge Shifting in a Polycarboxybetaine Ester Gene Delivery Platform

Polycarboxybetaine esters (PCB-esters) can condense plasmid DNA into nanosized polyplexes for highly effective gene delivery with low toxicity. The design and characterization of tertiary CB-ester monomers and PCB-ester polymers are presented here to study the effects of molecular variation on functions important to nonviral gene transfer. Both buffering capacity and charge-shifting behavior can be tuned by modifying the distance between the charged groups and the ester size or type. A carbon spacer length (CSL) of one was found to bring the p<i>K</i>_a of the tertiary amine into the optimal range for proton buffering. Ester hydrolytic degradation switches this polymer from cationic (DNA binding) to zwitterionic (DNA releasing) form while conferring nontoxicity. To allow rapid and externally controlled degradation, the effect of this charge-switching behavior on DNA release from polyplexes was directly studied with a novel photolabile PCB-nitrobenzyl ester (PCB-NBE). Photoinitiated ester degradation precipitated the rapid release of 72 ± 5% of complexed DNA from PCB-NBE polyplexes. These insights reveal the key parameters important for the PCB-ester platform and the significance of charge switching to an effective and nontoxic nonviral gene delivery platform

table_1.PDF

<p>Disorders of sex development (DSD) consist of a wide range of conditions involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. GATA4 gene variants, mainly related to congenital heart defects (CHD), have also been recently associated with 46,XY DSD. In this study, we characterized three individuals presenting with 46,XY DSD with or without CHD and GATA4 variants in order to understand the phenotypical variability. We studied one patient presenting CHD and 46,XY gonadal dysgenesis, and two patients with a history of genetically unsolved 46,XY DSD, also known as male primary hypogonadism. Mutation analysis was carried out by candidate gene approach or targeted gene panel sequencing. Functional activity of GATA4 variants was tested in vitro on the CYP17 promoter involved in sex development using JEG3 cells. We found two novel and one previously described GATA4 variants located in the N-terminal zinc finger domain of the protein. Cys238Arg variant lost transcriptional activity on the CYP17 promoter reporter, while Trp228Cys and Pro226Leu behaved similar to wild type. These results were in line with bioinformatics simulation studies. Additional DSD variations, in the LRP4 and LHCGR genes, respectively, were identified in the two 46,XY individuals without CHD. Overall, our study shows that human GATA4 mutations identified in patients with 46,XY DSD may or may not be associated with CHD. Possible explanations for phenotypical variability may comprise incomplete penetrance, variable sensitivity of partner genes, and oligogenic mechanisms.</p

table_2.PDF

<p>Disorders of sex development (DSD) consist of a wide range of conditions involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. GATA4 gene variants, mainly related to congenital heart defects (CHD), have also been recently associated with 46,XY DSD. In this study, we characterized three individuals presenting with 46,XY DSD with or without CHD and GATA4 variants in order to understand the phenotypical variability. We studied one patient presenting CHD and 46,XY gonadal dysgenesis, and two patients with a history of genetically unsolved 46,XY DSD, also known as male primary hypogonadism. Mutation analysis was carried out by candidate gene approach or targeted gene panel sequencing. Functional activity of GATA4 variants was tested in vitro on the CYP17 promoter involved in sex development using JEG3 cells. We found two novel and one previously described GATA4 variants located in the N-terminal zinc finger domain of the protein. Cys238Arg variant lost transcriptional activity on the CYP17 promoter reporter, while Trp228Cys and Pro226Leu behaved similar to wild type. These results were in line with bioinformatics simulation studies. Additional DSD variations, in the LRP4 and LHCGR genes, respectively, were identified in the two 46,XY individuals without CHD. Overall, our study shows that human GATA4 mutations identified in patients with 46,XY DSD may or may not be associated with CHD. Possible explanations for phenotypical variability may comprise incomplete penetrance, variable sensitivity of partner genes, and oligogenic mechanisms.</p

Analysis of <i>CD177</i> variations and <i>CD177P1</i> divergence from gDNA and cDNA sequence.

<p><b>A</b>. CD177^neg and CD177^hi cells were sorted from an individual who predominantly express CD177^hi in neutrophils (<i>left</i>). Sequencing traces showed <i>CD177</i> codon variations at indicated loci, comparing results from gDNA deep sequencing (top), CD177 mRNA isolated from purified CD177^hi cells (middle) and CD177^neg neutrophils (bottom). The reference nucleotide sequences were labelled in colour letters below. The two SNPs present in gDNA and cDNA of both cell subsets were labelled (*). <b>B</b>. A schematic summary of the variations in gDNA and mRNA in neutrophils of this subject. Sequence variations were indicated on the top of the gene/transcript and exons were indicated (E2, E4 etc) below. <i>CD177P1</i> nucleotides in exon 4, 5 and 7 were outlined. CD177P1 transcripts were shown in grey signifying expected NMD with the stop codon variation g.7497T in red.</p

<i>CD177</i> g.7497A allele frequencies correlate to neutrophil CD177 expression.

<p><b>A—C</b>. Association of reference allele frequency (g.7497A) with neutrophil CD177 expression measured by geometric mean fluorescence intensity (MFI) of CD177 on the cell surface of total neutrophils (<b>A</b>), percentages of CD177^hi neutrophils (<b>B</b>), and CD177^neg neutrophils (<b>C</b>) in the blood of cohort 2. <b>D</b>. Heat map of variant allele frequency (determined by deep sequencing) and neutrophil phenotypes (determined by flow cytometry) in cohort 1.</p

CD177 expression in neutrophils.

<p><b>A.</b> Neutrophils of 5 subjects were gated on CD66b⁺ cells and analysed for surface expression of CD177. Three neutrophil subsets defined by CD177 expression were shown: negative (neg) in black, intermediate (int) in orange and high (hi) in pink, percentages of each subset were indicated as numbers in the same colour. <b>B</b>. Atypical CD177 expression with CD177^int subset >20% of neutrophils. <b>C.</b> A dot plot showing percentages of CD177^hi versus CD177^neg of total neutrophils in healthy subjects, each dot represents one subject. Three major phenotypic groups were marked as CD177^hi, CD177^hi/neg, and CD177^null. <b>D.</b> Contour plots showing neutrophils from a CD177^null subject (left) and a tri-model CD177 expressing donor (right), co-stained with two CD177 specific monoclonal antibodies (MEM-166 and REA258).</p

<i>CD177</i> and <i>CD177P1</i> variations.

<p><b>A</b>. <i>CD177</i> locus on human chromosome 19 and a schematic comparison of <i>CD177</i> and <i>CD177P1</i> genes. <b>B.</b> Current annotation of three copy number variations of <i>CD177</i> and <i>CD177P1</i> gene polymorphisms. <b>C</b>. <i>CD177</i> reference allele frequencies of two polymorphisms, g.1991C in exon 4 and g.7497A in exon 7, of cohort 1. Each dot represents one of 40 tested subjects. 15 out of 40 subjects displayed allele frequencies of g.1991C and g.7497A simultaneously at 50%. 14/40 subjects harboured similarly 50% g.7497A allele but 75% g.1991C. <b>D</b>. Proposed <i>CD177/CD177P1</i> haplotypes in two loci of exon 4 (C/G) and exon 7 (A/T). <i>CD177</i> gene in black line and <i>CD177P1</i> in grey. The most frequent genotype is highlighted.</p

Two exons of enriched SNP density and a novel stop codon variation in <i>CD177</i> gene.

<p><b>A</b>. Summary of deep sequencing of <i>CD177</i> gene (located at 43.8Mb, band q13.2, of the forward strand of chromosome 19 in human genome 19, 43.3 Mb and q13.31 for hg38). Amplicons covering the entire coding sequence of 9 exons were sequenced. 100% coverage was obtained at indicated read depths. 24 low frequency SNPs were found in exon adjacent non-coding regions and 17 polymorphisms were identified in <i>CD177</i> coding sequences. Representative allelic frequencies of nine SNPs in exon 5 and 7 are displayed according to CD177 phenotypes. Graphs were generated from Integrative Genomics Viewer from Broad Institute referenced on hg19. <b>B</b>. SNV frequency of 41 variants (called from hg19) in cohort 1. Each dashed line represents genotypes from a single individual. A schematic <i>CD177</i> gene structure is shown of variants within <i>CD177</i>. <b>C</b>. Sanger sequencing of <i>CD177</i> exon 7 in DNA isolated from neutrophils (upper panel) and saliva (lower panel) from three individuals same as shown in <b>A</b>. Reference (Ref) and variant (Var) read frequencies derived from deep sequencing and CD177 neutrophil phenotypes for each subject were indicated on the top of each panel.</p

Ectopic and allelic <i>CD177P1</i> exon 7 conversion.

<p><b>A</b>. CD177^hi and CD177^neg neutrophils were sorted from a single donor with bimodal CD177 expression (<i>left</i>). Genomic variant allele frequencies were determined by deep sequencing (<i>top</i>), and compared with sequence variations in cDNA from CD177^hi (<i>middle</i>) and CD177^neg neutrophils (<i>bottom</i>). Two <i>CD177</i> transcripts were found in CD177^neg neutrophils. SNPs present in gDNA and cDNA of CD177^neg subsets but absent from CD177^hi cells are labelled (*). <b>B</b>. Schematic summaries of <i>CD177</i> vs <i>CD177P1</i> gDNA variations and CD177 mRNA in two neutrophil subsets. <i>CD177P1</i> derived nucleotides in exon 4, 5 and 7 are outlined, suggesting one <i>CD177</i> allele partially supplied by <i>CD177P1</i> exon 7. <b>C</b>&<b>E</b>. Genomic sequence traces in indicated loci within exon 4, 5 and 7 of two CD177^null individuals. Only <i>CD177P1</i> exon 7 sequences are detected in CD177^null subjects, who harbour both <i>CD177</i> and <i>CD177P1</i> upstream elements, i.e., exon 4 (<b>C</b>) and exon 5 (<b>E</b>). <b>D</b>&<b>F</b>. Schematic genomic <i>CD177</i>/<i>CD177P1</i> structures of CD177^null individuals as shown in <b>C</b>&<b>E</b>. <b>G</b>. Confirmation of ectopic and allelic <i>CD177P1</i> exon 7 conversion in three subjects by MLPA. The plots show the peak ratio of probes for indicated loci of <i>CD177</i> and <i>CD177P1</i> genes. Exon 2 is used as a reference read out of the <i>CD177</i> gene only; probes for exon 4, 5, 7 and 9 bind to both <i>CD177</i> and <i>CD177P1</i>, whereas probes labelled as (P1) are specific to <i>CD177P1</i> exon 5 and 7. The graph shows one copy duplication of <i>CD177P1</i> exon 7 in blue in the same subject as shown in <b>A</b> & <b>B</b>; and two copies duplication (allelic conversion) in the two CD177^null subjects (red & orange) as shown in <b>C-F</b>. The subject in red also shows duplication of <i>CD177P1</i> exon 5 in concordant to <b>C</b> & <b>D</b>.</p