Statistical shape modelling to analyse the talus in paediatric clubfoot.

One fifth of idiopathic clubfoot deformities cannot be fully corrected by Serial Ponseti casting and deformity recurs in 20%-30% of cases. To avoid x-ray exposure, the joints with largely unossified bones are diagnosed with magnetic resonance images (MRI). Typically, geometric measurements are made in the MRI planes; however, this method is inaccurate compared to measurements on three-dimensional (3D) models of the joint. More accurate measurements using the 3D bone shapes may be better at identifying differences between groups; and therefore, improve diagnosis. The entire set of shape features from MRI can be analysed simultaneously through statistical shape modelling (SSM) which assesses bone morphology of clubfoot in a more sensitive way. A method for SSM of the talus is developed in this study and the shape of the normal talus is compared with the one in clubfeet with residual deformity through both geometric measurements and SSM. Significant differences between two groups were found by both methods; and therefore, might contribute to improve diagnosis of clubfoot.

Residual Equinus After the Ponseti Method: An MRI-based 3-Dimensional Analysis.

BACKGROUND: Residual equinus deformity is present in up to 20% of clubfeet treated by the Ponseti method. These patients may require surgical release to restore dorsiflexion. Despite complete posterior release; persistent intraoperative equinus may be present and suggest concurrent joint incongruity. The purpose of this study was to characterize differences in ankle morphology in toddlers with residual equinus following the Ponseti method. METHODS: Preoperative magnetic resonance imaging (MRI) data from 10 patients who underwent reconstruction (17 feet; 7 bilateral, 3 unilateral clubfeet) for persistent equinus were compared with 16 age-matched controls. Through reverse engineering software, MRI data were used to generate 3-dimensional (3D) models. Four talus-based measures were performed on both MRI data and 3D models-neck depth, neck angle, width, and length. Models were also used to calculate talus volume and arc of curvature (plafond and talar dome). Standard statistical analyses were performed. RESULTS: Talus volumes, width, and length were less in clubfeet then in control feet. Although some measures were significant there was no mismatch with the ankle mortise dimensions or arc curvature that could account for any decrease in dorsiflexion. We found that from MRI measures the clubfoot neck depth was 2.3 versus 3.6 mm in controls (P<0.001) and from 3D modeling the clubfoot neck depth was 2.3 and 3.5 mm in controls (P=0.003). With 3D modeling talus clubfoot neck angle was 153.7 versus 140.4 degrees in controls (P=0.01). The clubfoot neck angle obtained from MRI measures were also different yet not significant [126.6 in clubfeet versus 122.5 degrees in controls (P=0.12)]. CONCLUSIONS: In comparison to age-matched feet; we have noted a decrease in talar neck depth and an obtuse talar neck angle in clubfeet treated in the manner of Ponseti. This may result in anterior ankle impingement and be the cause of residual equinus despite posterior release. In these procedures, the surgeon should recognize this possibility when the amount of dorsiflexion is less than expected. LEVEL OF EVIDENCE: Level III-case control study.

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors.

It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

Development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors.

The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previously developed an RCL assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and human cells as RCL amplification/indicator cells. Here we report an additional RCL assay that tests for the presence of theoretical "equine-tropic" RCLs. This approach provides further assurance of safety by detecting putative RCLs with an equine cell-specific tropism that might not be efficiently amplified by the human cell-based RCL assay. We tested the ability of accessory gene-deficient EIAV mutant viruses to replicate in a highly permissive equine cell line to direct our choice of a suitable EIAV-derived positive control. In addition, we report for the first time the mathematical rationale for use of the Poisson distribution to calculate minimal infectious dose of positive control virus and for use in monitoring assay positive/spike control failures in accumulating data sets. No RCLs have been detected in Good Manufacturing Practice (GMP)-compliant RCL assays to date, further demonstrating that RCL formation is highly unlikely in contemporary minimal lentiviral vector systems.

Generation of cell lines to complement adenovirus vectors using recombination-mediated cassette exchange.

BACKGROUND: Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. RESULTS: We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. CONCLUSIONS: RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression.

Factors that influence VSV-G pseudotyping and transduction efficiency of lentiviral vectors-in vitro and in vivo implications.

Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.

Activation of the early-late switch in adenovirus type 5 major late transcription unit expression by L4 gene products.

The adenovirus major late transcription unit (MLTU) encodes multiple proteins from five regions, L1 to L5, through differential splicing and polyadenylation. MLTU expression is temporally regulated; only a single product from L1 (52/55K) is expressed prior to replication, but a subsequent switch, the mechanism of which has not been defined, leads to full expression that encompasses L1 IIIa and all L2 to L5 products. Transfection of a plasmid containing the complete MLTU gave a full array of proteins in proportions similar to those in a late infection, and in a time course, the temporal pattern of expression in a natural infection was reproduced. However, a plasmid truncated after the L3 poly(A) site exclusively expressed the L1 52/55K protein and was defective in the switch to full gene expression from L1 to L3. The L4 33K protein, supplied in trans, was sufficient to upregulate cytoplasmic mRNA for MLTU products characteristic of the late pattern of expression to levels comparable to those produced by the full-length MLTU. There was a corresponding increase in expression of the L1 IIIa, L2, and L3 proteins, except hexon. Hexon protein expression additionally required both the L4 100K protein in trans and sequences downstream of the L3 poly(A) site in cis. These results indicate that induction of L4 protein expression is a key event in the early-late switch in MLTU expression, which we propose is precipitated by small amounts of L4 expression in a feed-forward activation mechanism.