Use of a bioamplification assay to detect nonselective recombinants and assess the genetic stability of oncolytic adenoviruses.

Detection of nonselective adenoviruses in tissue- or tumor-selective oncolytic adenovirus preparations presents a technical challenge because of the conditionally replication-competent nature of oncolytic adenoviruses. Although quantitative PCR has been used extensively for detecting specific genes that are likely present in nonselective recombinants, the actual biological activity of nonselective genetic recombinants has not been demonstrated. Therefore, a bioassay that amplifies nonselective adenoviruses through multiple passages in nonpermissive cells was developed to detect biologically active nonselective recombinants using CG7870, a prostate-specific oncolytic adenovirus. The assay was sensitive, and its results were consistent with a quantitative PCR assay for four lots of CG7870. CG0070, a pan-tumor oncolytic adenovirus with no detectable wild-type-like recombinants by PCR, was subjected to a variation of this bioamplification assay using two different nonpermissive cell lines to both verify PCR results and assess its genetic stability under selection pressure. No evidence of the presence of biologically active nonselective recombinants was seen in the original material or after serial passaging in nonpermissive cells. Thus, this bioamplification assay is able to detect nonselective recombinants, and its results are consistent with quantitative PCR assays. A modified version of this assay is also useful for assessing the genetic stability of oncolytic adenoviruses that have no PCR-detectable recombinants.

Validation of cell density and viability assays using Cedex automated cell counter.

A method using Cedex automatic cell counter (Innovatis) to determine the cell density and viability of a whole cell-based immunotherapy product has been developed and validated for the assay performance characteristics including specificity, accuracy, precision, linearity, range, and robustness. Instrument-to-instrument variation due to intrinsic differences in handmade flow cells was also evaluated. For cell density, Cedex demonstrated acceptable specificity, accuracy and precision for cell densities ranging from 3.13x10(5) to approximately 1.0x10(7)cells/mL, with intermediate precision of about 5% relative standard deviation (RSD). However, a marked difference was observed between the two instruments studied and they therefore could not be used interchangeably without additional calibration procedures that went beyond the manufacturer's recommendation. For viability, mixing known numbers of non-viable cells with highly viable cells allowed evaluation of the specificity, accuracy and linearity of the viability determination. Acceptable levels of accuracy (95.3-106.4% recovery) and precision (RSD<5%) were demonstrated for the viability range from 50 to 100%. The instrument-to-instrument difference was less than 4.6%. The assays for both cell density and viability were sufficiently robust for assay parameters. However, the effect of certain parameters was cell line-dependent, suggesting that Cedex performance should be verified for each cell line of interest.

Cortical and thalamic axon pathfinding defects in Tbr1, Gbx2, and Pax6 mutant mice: evidence that cortical and thalamic axons interact and guide each other.

During development, cortical areas establish precise reciprocal projections with corresponding thalamic nuclei. Pioneer axons from the cortex and thalamus first meet in the intermediate zone of the subcortical telencephalon (subpallium). Their close interactions in the subpallium suggest that they may use each other for guidance. To test this hypothesis, the development of corticothalamic and thalamocortical connections was studied in mice with mutations of transcription factor genes expressed specifically in the cortex (Tbr1), the dorsal thalamus (Gbx2), or both (Pax6). In Tbr1 mutants, cortical pioneer axons entered the subpallium at the appropriate time, but most stopped growing without entering the diencephalon. Surprisingly, thalamic axons (which do not express Tbr1) deviated into the external capsule and amygdala regions, without entering the cortex. Conversely, in most Gbx2 mutants, thalamic axons were reduced in number and grew no farther than the subpallium. Cortical axons (which do not express Gbx2) grew into the subpallium but did not enter the diencephalon. In one Gbx2- /- case, sparse thalamocortical and corticothalamic projections both developed, but in no case did one projection reach its target and not the other. In Pax6 mutants, neither corticothalamic nor thalamocortical axons reached their targets. These results suggest that thalamocortical and corticothalamic projections may not form independently. After reaching the subpallium, each projection may require a molecularly intact reciprocal projection for further guidance. This type of mechanism ensures that thalamocortical and corticothalamic axons project reciprocally. However, the exact nature of the interaction between cortical and thalamic pioneer axons remains to be elucidated.