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High tibial osteotomy (HTO) is an effective surgical treatment for isolated medial compartment knee osteoarthritis; however, widespread adoption is limited due to difficulty in achieving the planned correction, and patient dissatisfaction due to soft tissue irritation. The aim of this study was to assess the accuracy of a novel HTO system with 3D printed patient specific implants and surgical guides using cadaveric specimens. Local ethics committee approval was obtained. The novel opening wedge HTO procedure was performed on eight cadaver leg specimens. Whole lower limb CT scans pre- and post-operatively provided geometrical assessment quantifying the discrepancy between pre-planned and post-operative measurements for key variables: the gap opening angle and the patient specific surgical instrumentation positioning. The average discrepancy between the pre-operative plan and the post-operative osteotomy correction angle was: 0.0 ± 0.2° The R

Regulatory T cells (Treg) critically maintain immuno-suppression in the tumor microenvironment, representing an attractive immuno-oncology target. The Treg lineage is defined by expression of the FOXP3 transcription factor, which controls immune-suppressive functions. We have developed the clinical candidate AZD8701, a next-generation antisense oligonucleotide inhibitor of FOXP3 (utilizing the Ionis Gen 2.5 cEt-modified ASO platform). AZD8701 treatment knocked down FOXP3 in primary human Tregs via free uptake (IC50 65nM), which was also associated with modulation of known FOXP3 target genes including 25-50% reduction in CTLA4, ICOS, CCR8 and GITR. Tregs treated with AZD8701 further exhibited reduced suppressive functions in in vitro suppression assays, which confirmed the functional effects of FOXP3 modulation. Finally, AZD8701 promoted dose-dependent knockdown of FOXP3 in humanized mice, including >50% FOXP3 knockdown at doses that can be feasibly achieved with the Gen 2.5 ASO platform in the clinic. To support the importance of FOXP3 in immuno-oncological settings, we characterized murine surrogate FOXP3 ASOs in the context of syngeneic tumour bearing mice. Murine FOXP3 ASOs similarly promoted >50% FOXP3 knockdown in mice and were well tolerated with no overt toxicological findings at high doses, over a maximum of 12 weeks of treatment. Murine FOXP3 ASOs significantly attenuated tumour growth in A20 and ID8-VEGF syngeneic models, which was associated with some complete tumour regressions. Moreover, we found that mouse surrogate FOXP3 ASOs promoted additive/enhanced therapeutic effects when combined with immune checkpoint blockade. Collectively, FOXP3 ASOs represent a first-in-class strategy to target Tregs in cancer in a highly selective manner. The clinical application of AZD8701 may provide therapeutic benefit to patients either as a monotherapy or in combination with immune checkpoint blocking agents. Citation Format: Charles Sinclair, Alexey S. Revenko, R B. Johnson, Alison Peter, Molly A. Taylor, Lisa A. Hettrick, Stephanie Klein, Anisha Solanki, Melissa Chapman, James Yates, Helen K. Angell, Andrew Watt, Danielle Gattis, Brett P. Monia, Simon T. Barry, Paul Lyne, Mark Edbrooke, Frederick Goldberg, A R. Macleod. Discovery and characterization of AZD8701, a high affinity antisense oligonucleotide targeting FOXP3 to relieve immunosuppression in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2713.

The eukaryotic translation initiation factor 4E (eIF4E) is a potent oncogene that is found to be dysregulated in 30% of human cancer, including colorectal carcinogenesis (CRC). ISIS 183750 is a second-generation antisense oligonucleotide (ASO) designed to inhibit the production of the eIF4E protein. In preclinical studies we found that EIF4e ASOs reduced expression of EIF4e mRNA and inhibited proliferation of colorectal carcinoma cells. An additive antiproliferative effect was observed in combination with irinotecan. We then performed a clinical trial evaluating this combination in patients with refractory cancer. No dose-limiting toxicities were seen but based on pharmacokinetic data and tolerability the dose of irinotecan was reduced to 160 mg/m(2) biweekly. Efficacy was evaluated in 15 patients with irinotecan-refractory colorectal cancer. The median time of disease control was 22.1 weeks. After ISIS 183750 treatment, peripheral blood levels of eIF4E mRNA were decreased in 13 of 19 patients. Matched pre- and posttreatment tumor biopsies showed decreased eIF4E mRNA levels in five of nine patients. In tumor tissue, the intracellular and stromal presence of ISIS 183750 was detected by IHC in all biopsied patients. Although there were no objective responses stable disease was seen in seven of 15 (47%) patients who were progressing before study entry, six of whom were stable at the time of the week 16 CT scan. We were also able to confirm through mandatory pre- and posttherapy tumor biopsies penetration of the ASO into the site of metastasis.

In contrast to other therapeutic approaches, the druggable universe is not limited with antisense technology, as these inhibitors can be rationally designed based on sequence information alone. Recent clinical data has demonstrated proof of mechanism and clinical benefit with antisense drugs across several disease areas including cancer. Together with recent advances in antisense chemistry, these findings suggest that this technology is poised to become an important therapeutic modality to inhibit both protein-coding RNAs as well as the more recently discovered non-protein coding RNAs and to help to bridge the existing pharmacogenomic divide in cancer drug discovery.

We present a technical note on 2 patients with post-traumatic symptomatic hyperextension of the knee treated with a new arthroscopic technique. Both patients were of similar ages with similar injuries resulting in an excess of hyperextension at the knee with resulting instability and pain. Both patients had not improved with a variety of nonoperative measures and 1 attempt each at simple arthroscopic debridement of the damaged tissue. Our technique involves carefully scarring the damaged posterior capsule arthroscopically, followed by extension block bracing for 12 weeks. In 2 patients who had not improved with previously described techniques, we achieved a correction of the excess hyperextension with resulting improvement in their symptoms. Two years after surgery, both patients had significantly improved Lysholm and Tegner activity scores and had returned to work. We believe this technique to be reliable and reproducible.

We have isolated a cDNA that contains the complete coding sequence of a 3.0 × 10 3 base human fibroblast mRNA together with a large part of its 3′ untranslated sequence. The deduced protein sequence is very similar if not identical to the sequence of horse platelet tropomyosin, a 247 amino acid protein. In vitro translation of an SP6 transcript of this cDNA reveals that the protein product of the 3.0 × 10 3 base mRNA is TM30 pl , one of the five proteins in human fibroblasts that have been shown to possess the physical and chemical characteristics of tropomyosin. This mRNA is encoded by a gene family that consists of a functional gene and multiple RNA-copy pseudogenes. This family of sequences is distinct from the gene family encoding TM30 nm , a cytoskeletal tropomyosin very similar in electrophoretic mobility to TM30 pl , but which shows significant differences in primary structure.

The long-term effects of colostrum on active immunity to two unrelated antigens are described. Lambs were fed with pooled colostrum—to equalise passive immunity—with or without added antibodies to egg albumin (Ea). There were significant breed differences in the response both to Brucella abortus measured at one month of age, and to Ea, measured at three months of age, although there was no significant correlation between the responses to the two antigens, either within or between breeds. Surprisingly, whereas antibodies to Ea caused a four-fold reduction in antibody production to B abortus , they did not affect the overall mean response to Ea. But the timing of the response to Ea was significantly affected, suggesting that the low persisting concentrations of antibody had caused qualitative changes in the response.

We have isolated clones of human genomic DNA which contain the structural elements of the hTMnm gene. In non-muscle tissue this gene produces a 2.5 kb (1 kb = 103 bases or base-pairs) mRNA encoding TM30nm, a 248 amino acid cytoskeletal tropomyosin. In muscle, alternative splicing of this gene results in the expression of a 1.3 kb mRNA encoding a 285 amino acid skeletal muscle α-tropomyosin. The hTMnm gene spans at least 42 kb of DNA and consists of 13 exons, only five of which are common to both the 2.5 kb and 1.3 kb transcripts. The boundaries of the exons giving rise to the muscle-specific isoform are identical to the base to those of other genes encoding muscle tropomyosins. A comparison of the structures of exons encoding the amino-terminal sequences of the muscle and non-muscle isoforms suggests that the hTMnm gene has evolved by a specific pattern of exon duplication with alternative splicing.

We have isolated and characterized a human genomic DNA sequence that defines a family of closely related sequences. At least one member of this family expresses a 2.5 X 10(3) base messenger RNA transcript encoding a 30,000 molecular weight tropomyosin in human fibroblasts. The coding sequence of this mRNA but not the non-coding sequence is also related to that of a 1.1 X 10(3) base mRNA encoding a 36,000 molecular weight non-muscle tropomyosin. This demonstrates the existence of at least two functional genes encoding human non-muscle tropomyosins.

Unc-54 I is the structural gene for a myosin heavy chain present in a major fraction of the total myosin of Caenorhabditis elegans. The allele e675, which possesses a normal amount of myosin but fails to assemble thick filaments, has been shown previously to contain a novel heavy chain of molecular weight 2 X 10(5), shorter by 10(4) than the wild-type (N2) unc-54 gene product. The structural alteration of the E675 heavy chain is an internal deletion of 10(4) molecular weight near the COOH terminus of the molecule. This has been determined by mapping the partial digestion products of heavy chain fragments labeled specifically at their NH2 termini.

Mutants in the unc -54 gene of Caenorhabditis elegans have been characterized by cyanylation and sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the total myosin present in each mutant. In the recessive mutants lacking a major fraction of the total myosin, the high molecular weight doublet of 15 × 10 4 and 14 × 10 4 which dominates the cyanylation pattern of the total wild-type myosin is absent. In the mutant E675, which possesses a novel heavy-chain with a molecular weight of 2 × 10 5 , each component of the cyanylation doublet is reduced by 10 4 daltons, indicating that the doublet is derived from partial cleavage of a single polypeptide chain. This suggests that unc -54 is the structural gene for a myosin heavy-chain present in a major fraction of the total nematode myosin.

The cytoskeletal extracts of cultured human fibroblasts were found to contain at least four distinct polypeptides, each of which demonstrated the resistance to denaturation and the acidic isoelectric point characteristic of tropomyosin. One of these, hscp 36 (heat-stable cytoskeletal protein having an apparent molecular weight of 36,000), cross-reacted efficiently with an antiserum to chicken skeletal muscle tropomyosin. Furthermore, the messenger RNA coding for hscp 36 was selected by a chicken complementary DNA clone containing a tropomyosin sequence. The abundance of mRNA coding for hscp 36 was found to be similar in both normal and simian virus 40 (SV40) transformed human fibroblasts. The apparent molecular weight of hscp 36 is different from non-muscle tropomyosins previously isolated from human sources, which show the apparent molecular weight of 30,000 normally associated with non-muscle tropomyosin. This, together with the complexity of the heat-stable cytoskeletal proteins present in human fibroblasts, suggests the existence of multiple genes coding for human non-muscle tropomyosins.

The trk oncogene is a human transforming gene generated by the fusion of tropomyosin gene sequences to a truncated tyrosine kinase receptor gene1. We have now characterized the normal tropomyosin gene from which the trk oncogene is derived. At least two different transcripts are expressed by this gene using a tissue-specific alternative messenger RNA splicing mechanism: a 2.5-kilobase (kb) mRNA encoding a 248-amino-acid tropomyosin in human fibroblasts and a 1.3-kb mRNA encoding a 285-amino-acid tropomyosin in human skeletal muscle. The rearrangement which generates the trk oncogene preserves most of the tropomyosin-coding sequences of the normal gene, including exons alternatively spliced in muscle and non-muscle tissue. We therefore expect the trk oncogene to show a tissue-specific pattern of transforming activity. Correct expression of the trk oncogene can occur only in non-muscle tissues. In muscle tissue the oncogene would almost certainly be inactive, as splicing according to the alternative muscle pattern aborts synthesis of the tryosine kinase domain.

Recombinant plasmids have been isolated which contain sequences complementary to two distinct alpha-tropomyosin mRNA species present in chicken leg muscle. The proteins coded for by these different mRNAs could be distinguished by their electrophoretic behaviour in the presence of 3.5 M urea. The properties of the minor alpha-tropomyosin of chicken leg muscle were similar to those reported for the alpha-tropomyosin of slow twitch chicken skeletal muscle. Sequence analysis of available plasmids showed that the deduced protein sequences of both types of alpha-tropomyosin were very similar and closely related to the known protein sequence of rabbit alpha-tropomyosin. However considerable variation in nucleotide coding sequence of the two alpha-tropomyosin mRNAs was found.

We have isolated a cDNA clone from a human fibroblast cDNA library that contains the entire protein-coding region of a 1.1-kilobase mRNA. This mRNA encodes a 284-amino acid tropomyosin, the primary structure of which most closely resembles smooth muscle tropomyosin. Thus, the expression of both 284-amino acid muscle-type and 247-amino acid non-muscle-type tropomyosins appears to be a normal feature of human non-muscle cells. We also present evidence to suggest that this cytoskeletal tropomyosin and a human skeletal muscle beta-tropomyosin are derived from a common structural gene by an alternative RNA splicing mechanism.

Recombinant plasmids have been constructed with contain sequences complementary to the mRNA coding for skeletal muscle alpha-tropomyosin. These recombinants were detected initially using a selective cDNA probe and subsequently using a messenger RNA selection assay. alpha-TM plasmids hybridize to a singly mRNA species smaller than 18S ribosomal RNA and found only in skeletal muscle. Cross-hybridization with mRNA's coding for other tropomyosins could not be detected under normal conditions. However, under conditions of reduced stringency alpha- TM plasmids cross-hydridize with an RNA species in heart muscle which may code for cardiac tropomyosin.

The expression of the mRNA coding for glyceraldehyde-3-phosphate dehydrogenase has been studied during embryonic development of the chicken. In each tissue examined, only one mRNA species coding for a single glyceraldehyde-3-phosphate dehydrogenase subunit could be detected using translation in vitro with RNA blotting and hybridization. The mRNA species coding for glyceraldehyde-3-phosphate dehydrogenase subunits in different chicken tissues have identical electrophoretic mobilities suggesting that they are structurally very similar if not identical.

We have determined the sequence of a 2.5 kb mRNA in human fibroblasts encoding a 248 amino acid cytoskeletal tropomyosin. The protein product of this mRNA is TM30nm, one of five tropomyosin-like proteins in human fibroblasts. The structural gene encoding this mRNA can also produce a 1.3 kb mRNA encoding a 285 amino acid skeletal muscle alpha-tropomyosin by tissue-specific alternative mRNA splicing. However, the multiple RNA-copy pseudogenes of this gene family are derived largely if not exclusively from transcripts processed according to the pattern observed in non-muscle cells.