Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development.

The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.

Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats.

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

A priA Mutant Expressed in Two Pieces Has Almost Full Activity in Escherichia coli K-12.

The ability to restart broken DNA replication forks is essential across all domains of life. In Escherichia coli, the priA, priB, priC, and dnaT genes encode the replication restart proteins (RRPs) to accomplish this task. PriA plays a critical role in replication restart such that its absence reveals a dramatic phenotype: poor growth, high basal levels of SOS expression, poorly partitioned nucleoids (Par-), UV sensitivity, and recombination deficiency (Rec-). PriA has 733 amino acids, and its structure is composed of six domains that enable it to bind to DNA replication fork-like structures, remodel the strands of DNA, interact with SSB (single-stranded DNA binding protein), PriB, and DnaT, and display ATPase, helicase, and translocase activities. We have characterized a new priA mutation called priA316::cat It is a composite mutation involving an insertion that truncates the protein within the winged-helix domain (at the 154th codon) and an ACG (Thr)-to-ATG (Met) mutation that allows reinitiation of translation at the 157th codon such that PriA is expressed in two pieces. priA316::cat phenotypes are like those of the wild type for growth, recombination, and UV resistance, revealing only a slightly increased level of SOS expression and defects in nucleoid partitioning in the mutant. Both parts of PriA are required for activity, and the N-terminal fragment can be optimized to yield wild-type activity. A deletion of the lon protease suppresses priA316::cat phenotypes. We hypothesize the two parts of PriA form a complex that supplies most of the PriA activity needed in the cell.IMPORTANCE PriA is a highly conserved multifunctional protein that plays a crucial role in the essential process of replication restart. Here we characterize an insertion mutation of priA with an intragenic suppressor such that it is now made in two parts. These two pieces split the winged-helix domain to separate the N-terminal 3' DNA-binding domain from the C-terminal domain of PriA. It is hypothesized that the two pieces form a complex that is capable of almost wild type priA function. The composite mutation leads to a moderate level of SOS expression and defects in partitioning of the chromosomes. Full function is restored by deletion of lon, suggesting that stability of this complex may be a reason for the partial phenotypes seen.

Phosphate is the third nutrient monitored by TOR in Candida albicans and provides a target for fungal-specific indirect TOR inhibition.

The Target of Rapamycin (TOR) pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans Eukaryotic Target of Rapamycin complex 1 (TORC1) induces growth and proliferation in response to nitrogen and carbon source availability. Our unbiased genetic approach seeking unknown components of TORC1 signaling in C. albicans revealed that the phosphate transporter Pho84 is required for normal TORC1 activity. We found that mutants in PHO84 are hypersensitive to rapamycin and in response to phosphate feeding, generate less phosphorylated ribosomal protein S6 (P-S6) than the WT. The small GTPase Gtr1, a component of the TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in Gtr1 but not in another TORC1-activating GTPase, Rhb1, are defective in the P-S6 response to phosphate. Overexpression of Gtr1 and a constitutively active Gtr1Q67L mutant suppresses TORC1-related defects. In Saccharomyces cerevisiae pho84 mutants, constitutively active Gtr1 suppresses a TORC1 signaling defect but does not rescue rapamycin hypersensitivity. Hence, connections from phosphate homeostasis (PHO) to TORC1 may differ between C. albicans and S. cerevisiae The converse direction of signaling from TORC1 to the PHO regulon previously observed in S. cerevisiae was genetically shown in C. albicans using conditional TOR1 alleles. A small molecule inhibitor of Pho84, a Food and Drug Administration-approved drug, inhibits TORC1 signaling and potentiates the activity of the antifungals amphotericin B and micafungin. Anabolic TORC1-dependent processes require significant amounts of phosphate. Our study shows that phosphate availability is monitored and also controlled by TORC1 and that TORC1 can be indirectly targeted by inhibiting Pho84.

Structural mechanisms of PriA-mediated DNA replication restart.

Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.

Physiologic expression of the Candida albicans pescadillo homolog is required for virulence in a murine model of hematogenously disseminated candidiasis.

Morphogenetic conversions contribute to the pathogenesis of Candida albicans invasive infections. Many studies to date have convincingly demonstrated a link between filamentation and virulence; however, relatively little is known regarding the role of the filament-to-yeast transition during the pathogenesis of invasive candidiasis. We previously identified the C. albicans pescadillo homolog (PES1) as essential during yeast growth and growth of lateral yeast on hyphae but not during hyphal growth. Furthermore, we demonstrated that PES1 is required for virulence in vivo in a Galleria mellonella larva model of candidiasis. Here, we have used a regulatable tetO-PES1/pes1 strain to assess the contribution of C. albicans PES1 to pathogenesis in the commonly used and clinically relevant murine model of hematogenously disseminated candidiasis. Our results indicate that a physiologically controlled level of PES1 expression is required for full virulence in this animal model, with virulence defects observed both when PES1 is overexpressed and and when it is depleted. The pathogenetic defect of cells depleted of PES1 is not due to a general growth defect, as demonstrated by the fact that PES1-depleted cells still kill Caenorhabditis elegans as efficiently as the wild type due to hyphal outgrowth through worm tissues. Our results suggest a critical role of lateral yeast growth in the ability of C. albicans to normally proliferate within tissues, as well as a pivotal role for Pes1 in the normal developmental cycle of C. albicans within the mammalian host during infection.

Regulated transcription of the Saccharomyces cerevisiae phosphatidylinositol biosynthetic gene, PIS1, yields pleiotropic effects on phospholipid synthesis.

Phosphatidylinositol is an important membrane lipid in Saccharomyces cerevisiae and other eukaryotes. Phosphatidylinositol and its metabolites (phosphoinositides, inositol polyphosphates, etc.) affect many cellular processes with implications in human diseases. Phosphatidylinositol synthesis in S. cerevisiae requires the essential PIS1 gene. Recent studies reveal that PIS1 expression is regulated at the level of transcription in response to carbon source, oxygen, and zinc. However, the consequence of this regulation on phosphatidylinositol levels and functions has not been thoroughly studied. To investigate this, we created a strain with a galactose-inducible GAL1-PIS1 gene. In this strain, the amount of phosphatidylinositol correlated with PIS1 expression but did not exceed c. 25% of the total phospholipid composition. Interestingly, we found that 4% phosphatidylinositol was sufficient for cell growth. We also found that reduced PIS1 expression yielded derepression of two phospholipid biosynthetic genes (INO1 and CHO1) and the INO2 regulatory gene. Consistent with this derepression, reduced PIS1 expression also yielded an overproduction of inositol (Opi(-)) phenotype. The effect on transcription of the INO1, CHO1, and INO2 genes is consistent with the accepted model that phosphatidic acid (PA) is the signal for regulation of these genes because decreased phosphatidylinositol synthesis would affect PA levels.

Transcription regulation of the Saccharomyces cerevisiae PIS1 gene by inositol and the pleiotropic regulator, Ume6p.

In Saccharomyces cerevisiae, transcription of most of the phospholipid biosynthetic genes (e.g. INO1, CHO1, CHO2 and OPI3) is repressed by growth in the presence of inositol and choline and derepressed in their absence. This regulation requires the Ino2p and Ino4p activators and the Opi1p repressor. The PIS1 structural gene is required for the synthesis of the essential lipid phosphatidylinositol. Previous reports show that PIS1 expression is uncoupled from inositol/choline regulation, but is regulated by carbon source, hypoxia and zinc. However, in this study we found that the expression of PIS1 is induced twofold by inositol. This regulation did not require Ino2p and Ino4p, although Ino4p was required for full expression. Ino4p is a basic helix-loop-helix protein that requires a binding partner. Curiously, none of the other basic helix-loop-helix proteins affected PIS1 expression. Inositol induction did require another general regulator of phospholipid biosynthesis, Ume6p. Ume6p was found to be a positive regulator of PIS1 gene expression. Ume6p, and several associated factors, were required for inositol-mediated induction and chromatin immunoprecipitation analysis showed that Ume6p directly regulates PIS1 expression. Thus, we demonstrate novel regulation of the PIS1 gene by Ume6p.

Phosphatidylinositol biosynthesis: biochemistry and regulation.

Phosphatidylinositol (PI) is a ubiquitous membrane lipid in eukaryotes. It is becoming increasingly obvious that PI and its metabolites play a myriad of very diverse roles in eukaryotic cells. The Saccharomyces cerevisiae PIS1 gene is essential and encodes PI synthase, which is required for the synthesis of PI. Recently, PIS1 expression was found to be regulated in response to carbon source and oxygen availability. It is particularly significant that the promoter elements required for these responses are conserved evolutionarily throughout the Saccharomyces genus. In addition, several genome-wide strategies coupled with more traditional screens suggest that several other factors regulate PIS1 expression. The impact of regulating PIS1 expression on PI synthesis will be discussed along with the possible role(s) that this may have on diseases such as cancer.