Patent Application: US-201415100638-A

Abstract:
the invention relates to the use of microrna 96 and precursors and mimics thereof for the inhibition of vascular cell proliferation and / or vascular remodelling , and for the treatment of associated medical conditions such as pulmonary arterial hypertension .

Description:
research suggests that micrornas ( mirs ) play a crucial role in the pathobiology of pulmonary arterial hypertension ( pah ). heritable pah ( hpah ) is associated with mutations in the gene for bone morphogenetic protein receptor 2 ( bmpr2 ). the inventors have studied a bmpr2 mouse model of pah carrying a mutation also found in pah patients , the bmpr2 r899x +/− mouse . in this model , gender emerges as a key underlying regulator of basal expression of mirs within pulmonary artery smooth muscle cells ( pasmcs ). mir - 96 was down - regulated in pasmcs from female bmpr2 r899x +/− mice compared to the female wild type ( wt ) whereas expression between the male transgenics and their wt controls was unchanged . global expression analysis showed highest mir - 96 expression within the lung . the target gene for the 5 - ht1b receptor was inversely correlated with the expression of mir - 96 within the female bmpr2 r899x +/− pasmcs at both mrna and protein level . the 5ht1b gene was validated as a target for mir - 96 by luciferase reporter assay . the inventors also verified that pasmc from female pah patients with bmpr2 mutations exhibit low mir - 96 and high 5ht1b expression . these results suggest sex hormones such as oestrogen may play a role in regulating mir - 96 . consistent with this we have shown that inhibiting the synthesis of endogenous oestrogen in vivo increased lung mir - 96 and decrease 5 - ht1b expression within female mice only . direct stimulation with oestrogen in human pasmcs caused a reduction in mir - 96 and increase in 5 - ht1b expression . in summary , a decrease in mir - 96 was associated with increased 5ht1b expression . this suggests that mir - 96 mimic / pre - mir - 96 strategies may be a novel therapeutic approach to pah . in support of this hypothesis , pre - clinical studies confirm that a mir - 96 mimic is effective in preventing or reversing pulmonary hypertension in mouse models of pulmonary hypertension . female and male pulmonary artery smooth muscle cells ( pasmcs ) derived from bmpr2 +/− r899x mice and corresponding wild type control mice were provided by prof . n . w . morrell ( university of cambridge , cambridge , uk ). bmpr2 +/− r899x mice have a heterozygous knock - in mutation of the bmpr2 gene . the mice were similar to those described by west et al . ( am j physiol lung cell mol physiol 295 : l744 - l755 , 2008 ). briefly pamscs were grown in 20 % fbs ( foetal bovine serum ) dmem ( dulbecco &# 39 ; s modified eagle medium ) in the presence of growth factors ( epidermal growth factor , basic fibroblast growth factor and insulin ) and antibiotic ( penicillin streptomycin ). pasmcs were utilised between passage 4 and 8 and cell viability was based on distinct smooth muscle cell morphology . female human pasmcs were provided by prof . n . w . morrell ( university of cambridge , cambridge , uk ). briefly human pasmcs were explanted from the small distal pulmonary arteries of the pulmonary vasculature from three pah patients , two with hpah and one with ipah . pasmcs derived from non - pah subjects were utilised as controls . human pasmcs were grown in 10 % fbs dmem in the presence of antibiotic antimycotic solution ( containing penicillin , streptomycin and amphotericin ) and were used between passage 4 and 8 . pasmc phenotype was confirmed via smooth muscle cell morphology . total rna was extracted using the mirneasy kit ( qiagen ) according to the manufacturer &# 39 ; s instructions and rna purity quantified using nanodrop - 1000 spectrophotometer . expression of mirs and mrna was assessed by taqman quantitative - pcr as previously described [ 17 ]. to obtain a fold change mir expression data was normalised to u6 for mouse and rnu48 for human . mrna expression data was normalised to gapdh . all pasmc mirna and mrna expression data is n = 6 in triplicate . protein was extracted using the ripa lysis method . the ripa buffer was supplemented with proteases inhibitors pmsf , soybean trypsin inhibitor and benzamidine . briefly supplemented ripa buffer was added to pasmcs which were then agitated on ice for 10 minutes . cell lysates were collected via scraping . protein expression data was normalised to alpha - tubulin and quantified using densitometry . all protein expression data is n = 4 in duplicate . to assess whether mir - 96 was involved in the pathology of pulmonary hypertension , the microrna - 96 mimic ( applied biosystems , # mc10422 , having the base sequence identical to mature mouse and human mir - 96 ) was administered intravenously via the tail vein once a week for 2 weeks using the maxsuppressor ™ in vivo rna - lancer ii delivery method . the mimic was prepared in the delivery reagent as per manufacturing instructions at a dose of 1 . 5 mg / kg per injection i . e . ˜ 30 ug per mouse per injection . negative microrna mimic ( applied biosystems ) and pbs dosed animals were used as controls . female wild type ( wt ) c57 / bl ( charles river ) mice aged 2 months old were exposed to 14 day hypobaric hypoxia ( 10 % o 2 , 550 mbar ) as described previously ( 18 ). mice maintained in normoxic conditions ( 21 % o 2 , 1000 mbar ) were studied as controls . measurements of right ventricular systolic pressure ( rvsp ), right ventricular hypertrophy ( rvh ) and pulmonary vascular remodeling were performed as previously described ( 3 , 4 , 10 ) the psi - check - 2 dual luciferase reporter vector ( promega ) was utilised for the reporter assay . briefly , a fragment of the 3 ′ utr ( untranslated region ) of the 5 - ht1b receptor gene was generated by polymerase chain reaction ( pcr ) from genomic mouse dna and was cloned via the in - fusion cloning method into the multiple cloning site of the psi - check - 2 vector at the xhoi and the noti restriction sites . the psi - check - 2 5 - ht1b vector was then sequenced to confirm there was no unwanted mutations within the 3 ′ utr region of the 5 - ht1b . lug of psi - check - 2 control vector or the psi - check - 2 5ht1b vector along with either pre - mir - 96 ( ambion ) or scrambled mir ( ambion ) were co - transfected into hela cells using lipofectamine 2000 ( invitrogen ) and optimem ( invitrogen ) for a total of 6 hours before being replaced with 10 % fbs dmem . the hela cells were left for 48 hours and the luciferase activity measured using the dual - glo luciferase assay system ( promega ) and detected by a luminometer . the renilla luciferase activity was normalised to the internal firefly luciferase activity and data expressed as a percentage of the internal control . values are expressed as mean ± standard error of the mean . a t - test or 2 - way anova followed by bonferroni &# 39 ; s post - hoc test was performed to evaluate the statistical significance between all groups where appropriate . a probability level of p & lt ; 0 . 05 was defined as being statistically significant . fig1 demonstrates that many mirs were differentially affected by gender , and mir - 96 was severely down regulated in female bmpr2 r899x +/− mice vs wild type controls but not males ; mir - 96 levels were lower in female bmpr2 r899x +/− mice vs male bmpr2 r899x +/− mice . lung expression of mir - 96 is extremely high compared with other tissue levels which would facilitate selective targeting of the lung ( fig2 ). we confirmed that the 5ht1b receptor is a target for mir96 using luciferase reporter assay ( fig3 ). decreased expression of mir96 was associated with an elevation in the target gene for the 5ht1b receptor in the female bmpr2 r899x +/− mice ( fig4 ). the decreased mir96 expression was rescued by bmp4 stimulation ( fig5 ). the same phenotype is observed in pasmcs from smad1 +/− mice ( fig6 ). results have been confirmed in human pasmcs ( fig7 ). of especial interest is that in a female patient with the r899x mutation , 5ht1b levels were high and mir96 expression extremely low ( fig7 ). we also examined the effect of oestrogen depletion with an aromatase inhibitor , anastrozole on the expression of both mir96 and the 5ht1b receptor . previously we have shown that anastrozole reverses pah in female mice and rats but not in males ( unpublished ). here we show that depletion of oestrogen in vivo with anastrozole increased mir96 expression and decreased 5ht1b expression in the female mouse lung but not the male ( fig8 ). in keeping with this , the mir - 96 gene has oestrogen - responsive regions and oestrogen itself decreased expression of mir - 96 and increased expression of the 5ht1b receptor in ( fig9 ). fig1 and 11 confirm that a mir - 96 mimic can both prevent and reverse experimental ph in a mouse model in vivo . our results suggest that increased oestrogen and bmpr2 haploinsufficiency are associated with decreased mir - 96 expression . mir - 96 normally represses the translation of the gene for 5ht1b and so the decrease in mir - 96 induces an increase in 5ht1b expression . this facilitates pasmc proliferation by serotonin and pulmonary vascular remodelling and contributes to the development of pah . while the invention has been described in conjunction with the exemplary embodiments described above , many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure . accordingly , the exemplary embodiments of the invention set forth are considered to be illustrative and not limiting . various changes to the described embodiments may be made without departing from the spirit and scope of the invention . all documents cited herein are expressly incorporated by reference . 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