Patent Abstract:
new gene therapy constructions and compositions are the subject of present invention . the gene therapy compositions consist in adeno - associated vectors which jointly express insulin and glucokinase genes . the new gene therapy constructions are useful for treatment of diabetes either in dogs or human beings .

Detailed Description:
the significance and potential impact of the gene therapy invention approach , consisting of co - expression of low levels of insulin together with the enzyme glucokinase in skeletal muscle , are potentially enormous . normalization of glycemia with a one - time intervention would result in a great improvement of patients &# 39 ; quality of life and prevention of severe and costly secondary complications of diabetes . the data disclosed in the present invention show that this is feasible and safe . it should be noted that , compared to other experimental therapeutic approach to diabetes , the strategy displayed in the invention is based on engineering skeletal muscle , a readily accessible tissue that do not require any invasive procedures to be manipulated . this is a considerable advantage over other approaches , such as engineering the liver or transplanting insulin - producing β - cells . it should also be pointed out that the gene therapy composition and the method disclosed herein have the advantage of not requiring immunosuppression , as diabetic subjects are naturally immunologically tolerant to insulin and glucokinase ; additionally , even basal ( low ) levels of expression of insulin and glucokinase may result in a dramatic improvement of the disease profile in terms of quality of life ( better glycemic control ) and reduction of insulin requirements . thus , the use of two genes acting synergistically on glycemic control potentially represents a major advance in the management of t1d and t2d diabetes worldwide . therefore , the present invention relates gene therapy compositions which comprise at least a first vector carrying and allowing the expression of insulin gene ( ins ) and at least a second vector carrying and allowing the expression of glucokinase gene ( gck ). as alternative , the gene therapy compositions of present invention comprise a single vectors carrying and allowing the expression of both genes ( ins and gck ) operatively linked . moreover , ins and / or gck genes can be , any of them independently , autologous or heterologous genes with regard to the species wherein are being expressed . in a particular embodiment of the gene therapy compositions of the invention are characterized by the vectors are adeno associated virus based vector . in another particular embodiment of the gene therapy composition disclosed in the present invention , the first vector contains the cds of seq id no . 1 or the cds of seq id no . 3 . in another particular embodiment of the gene therapy composition disclosed in the present invention , the second vector contains the cds of seq id no . 2 or the cds of seq id no . 4 . in another particular embodiment of the gene therapy composition , the first and the second carrying gene vectors are the same . in another particular embodiment of the gene therapy composition , comprises a first vector containing the cds of seq id no . 1 and a second vector containing the cds of seq id no . 2 . in another particular embodiment of the gene therapy composition disclosed herein , the first vector is aav - ins and the second vector is aav - gck . in another particular embodiment , the gene therapy composition of the invention comprises a first vector containing the cds of seq id no . 3 and a second vector containing the cds of seq id no . 4 . in another particular embodiment of the gene therapy composition disclosed in the present invention , the first vector is aav - mhins and the second vector is aav - mhgck . in another particular embodiment , the gene therapy composition of the invention comprises a first vector containing the cds of seq id no . 1 or the cds of seq id no : 3 and a second vector containing the cds of seq id no . 2 or the cds of seq id no : 4 . in another particular embodiment , the gene therapy composition of the invention is characterized by the first vector is selected from aav - ins or aav - mhins and the second vector is selected from aav - gck or aav - mhgck . present invention also relates gene therapy compositions for use in the treatment of diabetes in mammals . in a particular embodiment of the gene therapy compositions disclosed herein , the mammal is a rodent , preferably mice , rats , gerbils and guinea pigs and more preferably mice and rats . in another preferred embodiment of the gene therapy compositions disclosed herein , the mammal is a dog . in another preferred embodiment of the gene therapy compositions disclosed herein , the mammal is a human being . present invention also disclosed a mutated human insulin ( mhins ) gene characterized by comprising the cds having seq id no : 3 and a mutated human glucokinase ( mhgck ) gene characterized by comprising the cds having seq id no : 4 . another object disclosed in the present invention is the mutated human insulin ( mhins ) gene , as disclosed previously , for use in the treatment of diabetes . present invention also disclosed the use of the mutated human insulin ( mhins ) gene disclosed herein for the manufacture of a medicament and / or a gene therapy composition for use in the treatment of diabetes . another object disclosed in the present invention is the mutated human glucokinase ( mhgck ) gene , as disclosed previously , for use in the treatment of diabetes . present invention also disclosed the use of the mutated human glucokinase ( mhgck ) gene disclosed herein for the manufacture of a medicament and / or a gene therapy composition for use in the treatment of diabetes . present invention also disclosed a method of treatment of diabetes which comprises the administration to a subject in need of it , of a therapeutically effective dose of a gene therapy composition according to the present invention . in a preferred embodiment of the invention , the method comprises the administration of the gene therapy composition disclosed herein , in a single dose for all the treatment . in another preferred embodiment of the invention , the method disclosed that the single dose is administered to muscle tissue by means of an unique multi - needle injection . present invention also disclosed a method of treatment of diabetes which comprises the administration to a subject in need of it , of a therapeutically effective dose of a gene therapy composition which comprises at least a vector carrying and allowing the expression of glucokinase gene ( gck ). in a preferred embodiment of the method of the present invention , the vector is an adeno - associated virus based vector . in another preferred embodiment of the method disclosed herein , the vector comprises the cds having either seq id no : 2 or seq id no : 4 . in another preferred embodiment of the method disclosed herein , the vector is selected from aav - mhgck or aav - gck . in another preferred embodiment of the method disclosed herein , the gene therapy composition is administered in a single dose for all the treatment . in another preferred embodiment of the method disclosed herein , the single dose is administered to muscle tissue by means of an unique multi - needle injection . in another preferred embodiment of the invention the method further comprises exogenous insulin injections . the invention will now be described in more detail by way of examples . the following examples are for illustrative purposes only and are not intended , nor should they be interpreted , to limit the scope of the invention . studies in diabetic beagle dogs used a unique 5 - point needle ( fig1 a ) to obtain widespread expression of a gfp reporter in skeletal muscle ( fig1 b ). subsequently , 2 . 5 × 10 12 vg / kg of aav1 - human ins was injected into dog 1 three days after diabetes induction with streptozotocin + alloxan ( 50 ). low levels of circulating human c - peptide were observed 4 days later , peaking after 2 weeks in association with hypoglycemia . dog 1 was sacrificed 21 days after treatment and strong insulin expression was detected in biopsies of the treated area ( fig1 c ). these results indicated that aav vectors injected in multiple sites can efficiently deliver the insulin gene to widespread areas and that aav - mediated gene transfer of insulin to a large animal model of diabetes was feasible , resulting in large amounts of insulin produced and secreted from the dog skeletal muscle . next goal of present invention was to determine the optimum dose able to achieve therapeutic efficacy without causing hypoglycemia . to this end , dog 2 was injected with 1 . 0 × 10 12 vg / kg of aav1 - human ins after diabetes induction . after gene transfer , fasting glycemia decreased to reach normoglycemia without becoming hypoglycemic ( fig2 a ). after ˜ 300 days , the fasting glycemia values became slightly hyperglycemic and have since remained stable . however , even when normoglycemic , we did not see a significant improvement in the ability of this dog to dispose glucose ( fig2 a ). this was despite detecting human c - peptide ˜ 70 days after treatment , with stable levels achieved after 130 days those have lasted for more than 800 days , suggesting the long - term potential of this treatment . muscle biopsies taken 14 and 270 days after treatment showed detectable insulin rna at both time points , whereas a pancreas biopsy at day 270 showed less than 10 % residual β - cell mass and no sign of regeneration . dog 2 demonstrated no adverse events , no signs of toxicity and had a normal weight gain profile suggesting that even modest levels of circulating insulin can have beneficial effects . dog 3 and dog 4 were made diabetic and treated with the same dose of aav1 - human ins as dog 2 and an equal dose ( 1 . 0 × 10 12 vg / kg ) of aav1 - rat gck . both dog 3 and 4 showed a more accelerated return to fasting normoglycemia ( fig2 b ). these dogs remained normoglycemic for a long period (& gt ; 2 years ). circulating human insulin and c - peptide levels in these dogs were detectable after treatment and , importantly , both dogs 3 and 4 showed an improved gtt profile compared with dog 2 ( fig3 b , c ). muscle biopsies 15 and 113 days after viral injection revealed strong expression of both insulin and gck , whereas a pancreas biopsy at 113 days confirmed & lt ; 5 % residual β - cell mass . no muscle damage was seen and , like dog 2 , we observed normal weight gain and no toxicity . together , these data suggests that the combined treatment with human ins and rat gck leads to more beneficial effects in terms of improvement of glycemic control ; these effects were not observed in dog 2 despite the expression of insulin . then experimental diabetes in dog 5 was induced and followed long - term progression of diabetes . despite the complete absence of exogenous insulin treatment , this dog showed a gradual return to fasted normoglycemia , also coinciding with summer times . about six months after diabetes induction , we observed a severe rise in glycemia ( fig4 a ) parallel with a strong decrease in body weight (& gt ; 30 %) and marked increase in liver transaminases ( fig4 b , c ). at that moment , dog 5 was treated with the same doses of aav1 - ins and aav1 - gck as dog 3 and 4 , which resulted in dramatic improvements of its metabolic profile . fasting glycemia dropped sharply within 30 days of treatment ( fig4 a ), coinciding with a rise in circulating human c - peptide and a persistent weight gain ( fig4 b ). biochemical signs of liver damage also normalized ( fig4 c ) and , most importantly , we observed an improved glucose disposal by gtt reminiscent of dog 3 and 4 ( fig3 d ). these results clearly demonstrate the beneficial effects of combined ins + gck therapy in long - term diabetic dogs . therefore , joint expression of insulin and gck in skeletal muscle is a safe approach that allows long - term survival in large diabetic animals (& gt ; 2 years ), body weight maintenance , normal physical performance and normalization of serum parameters . construction of mutated vectors for efficient expression of human insulin and human glucokinase the coding sequence of either human insulin gene ( hins ), containing specific sites for furin processing ( 36 ), or human glucokinase gene ( hgck ) was modified to obtain codon mutated sequences ( mhins or mhgck , respectively ) following geneart procedures ( 48 ). geneart process involves avoiding cis - acting sequence motifs as : internal tata - boxes , chi - sites and ribosomal entry sites ar - rich or gc - rich sequence stretches rna instability motifs repeat sequences and rna secondary structures ( crytic ) splice donor and acceptor sites in higher eukaryotes the codon usage was adapted in geneart process to codon bias of mus musculus genes . in addition , regions of very high (& gt ; 80 %) or very low (& lt ; 30 %) gc content were avoided when possible . the mutated gene constructs obtained showed cai ( codon adaptation index ) of 0 . 96 what means high and stable expression rates in mus musculus . gc - content adjustment made by the process of genart , prolongs mrna half - life of the mutated construct achieved . the mutated human insulin and gck genes described herein are then called mutated human genes . the mutated insulin and gck cdna was cloned in the multicloning site of the paav - mcs plasmid ( stratagene ; fig5 ) resulting in the plasmids paav - mhins and paav - mhgck respectively . this plasmid contains the cmv promoter and polya signal from growth hormone flanked by the two inverted terminal repeats ( itr ) of aav2 . itr sequences are required for packaging of the aav genome into the aav capsid , and are required for replication of the aav genome during aav production . adeno - associated vectors were generated by triple transfection of human embryonic kidney 293 cells ( hek293 ) cells according to standard methods . hek293 are cells from human origin that are stable transfected with the adenovirus e1 gene . the adenovirus e1 gene is required for adenovirus replication and also acts as a helper gene for aav replication . the invention uses hek293 cells for several purposes : 1 .— aav production using triple transfection method . for aav production , it is required to have the cassette of expression flanked by itr ( plasmid 1 ), a plasmid coding for rep and cap genes from the aav ( plasmid 2 ; provides replication functions for aav genome and the capsid proteins depending on the desired serotype ), a third plasmid coding for the essential genes of adenovirus required to provide helper function and support replication of aav ( plasmid 3 , also named as adenovirus helper plasmid which code for e2 , e4 and va genes ). in addition to e2 , e4 and va , e1 gene is necessary for replication of aav , in this case e1 gene is provided by the hek293cells instead of being in the adenovirus helper plasmid . 2 .— for dna transfection . the inventors have used hek293 to study expression , processing and secretion of insulin and expression of gk because they are very efficiently transfected with plasmid using calcium phosphate method . 3 .— hek293 cells were also used to study expression , processing and secretion of insulin and expression of gk from aav1 vectors , because this cell line ( and not others ) are permissive for aav1 - transduction . cells were cultured in roller bottles ( rb ) ( corning , lowell , mass .) in dmem 10 % fbs to 80 % confluence and co - transfected with a plasmid carrying the expression cassette flanked by the viral itrs ( described above ), a helper plasmid carrying the aav rep2 and cap1 genes , and a plasmid carrying the adenovirus helper functions ( both plasmids kindly provided by k . a . high , children &# 39 ; s hospital of philadelphia ). vectors were purified with an optimized method based on two consecutives cesium chloride gradients ( 49 ), dialyzed against pbs , filtered , titred by qpcr and stored at − 80 ° c . until use . hek293 cells were transfected with paavmhins and paavmhgck using calcium phospate standard method . for experiments using aav vectors , hek293 cells were infected with aav1mhins and aav1mhgck at different moi ( i . e . 10e4 , 10e5 , 10e6 vg / cell ). two days after transfection , cells were lysated with 1 ml of tripure ( roche ) and total rna was extracted with rnaeasy mini kit ( qiagen ). a northern blot was performed with 10 ug of rna and hibridized with the mhins ( cds of seq id no : 3 ) or the mhgck ( cds of seq id no : 4 ) cdna , respectively ( fig6 ). since these plasmids showed a high expression level of the gene of interest , adenoassociated type 1 viral vectors carrying these constructs were produced . subsequently , aav vectors were tested for their mrna expression in hek293 cells 96 h after transduction . high levels of transgene expression were detected by northern blot both with aav1 - mhins and aav1 - mhgck ( fig7 ). in addition to increased rna expression , the present invention has also detected a substantial increase in mhgck protein production by the mutated construct ( fig8 ). codon mutated human gck construct produce 600 % more protein than the rat gck construct and 300 % more protein than the human gck transgene (= non codon mutated ). this data , together with data disclose in example 3 ( fig6 and 7 ) of the present invention demonstrate that mhgck contruct result in higher rna and protein production compare with construct carrying rgck or the wild type human gck gene . to demonstrate functionality of these novel constructs , aav1 vectors coding for rat gck ( rgck , nm — 012565 ), wild type human gck ( hgck , nm — 033507 ) or codon mutated human gck ( mhgck , cds of seq id no : 4 ) were produced as disclosed in the previous example 3 and 4 . hek293 cells were transduced with the 3 different vectors and gck activity was measured . as shown in fig9 , the gck activity of codon mutated ( mhgck ) construct was higher than wild type human ( hgck ) construct and rat gck ( rgck ) contruct . to provide in vivo evidences of gck function , the inventors injected aav1 vectors coding for rgck , hgck and mhgck into 3 different muscles in the hindlimbs of healthy mice . one month after the injection these muscles were harvested and analyzed for gck activity . as shown in fig1 , muscles treated with mhgck vectors disclosed higher gck activity compare with hgck and rgck . these results clearly demonstrated superior effect of aav1 - mhgck vectors vs aav1 - rgck or aav1 - hgck and suggested that lower doses of codon mutated insulin vectors will be required to achieve same therapeutic effect than non - mutated vectors . mutated construct showed an in vitro and in vivo increased insulin and c - peptide production compare to standard vectors we aimed to compare the ability of the mutated insulin gene versus the non mutated insulin gene to produce human c - peptide and human insulin production . to this end , we transduced hek293 cells with two different adenoassociated vectors ( aav1mhins ) at 3 different mois ( 10e4 , 10e5 and 10e6 vg / cell ). four wells per moi and vector were used . two days after the infection , standard culture media ( dmem + 10 % fbs ) was changed to a serum - free media to avoid the ria detection of the media containing insulin . next day ( three days after the infection ) medium was collected and was analyzed by ria for the human c - peptide and insulin quantification . then it was observed a significant increase in human c - peptide levels ( fig1 ) and human insulin levels ( fig1 ) in aav1 - mhins treated cells compared with standard insulin construct ( aav1 - hins ). these data demonstrate that mutated insulin construct is more efficient in protein production and secretion that standard insulin gene . to provide in vivo evidences of increased insulin and c - peptide production between aav1 - mhins vs aav1 - hins vectors , healthy mice were injected in hindlimb muscles with a total dose of 1 , 4e11 vg / mouse . glycemia and insulinemia was measured two weeks after viral injection . as shown in fig1 , a significant reduction in fed glycemia was observed in animals injected with aav1 - mhins compare with aav1 - hins . in agreement with this , insulinemia ( fig1 a ) and c - peptide ( fig1 b ) was higher in aav1 - mhins treated mice . the data disclosed in the present invention , clearly demonstrated a superior effect of aav1 - mhins vectors vs aav1 - hins and suggested that lower doses of codon mutated insulin vectors will be required to achieve same therapeutic effect than non - mutated vectors ( hins ). the use of lower doses of vectors may have several advantages for gene therapy : a ) potential immunological responses might be reduced since it has been suggested that immunological responses to aav are dose dependent , b ) lower number of injection sites to distribute the insulin vector will be required . c ) vector manufacture demand will be lower . the present invention tested the efficacy of a combined gene therapy approach with aav1 vectors carrying codon mutated human constructs in diabetic mice . to this end , we injected aav1 - mhgck vectors , aav1 - mhins or both ( 10e12 vg per vector / kg ) into the hindlimbs of c57b16 diabetic mice . experimental t1d was induced by streptozotocin ( stz ) administration as in ( 36 ) and viral vectors were injected 15 days after stz . a control group of stz - treated mice was injected with aav1 - null vectors ( same vector capsid but without expression of any transgene ). animals treated with a combination of aav1 - mhins + aav1 - mhgck showed significant reduction in blood glucose levels both in fasted and fed conditions ( fig1 and 16 , respectively ) compared with aav1 - null vector - treated mice or single treatment with aav1 - mhins or aav1 - mhgck . combined therapy : gene therapy with aav1 - mhgck + exogenous insulin in t1d and t2d the present invention have also evaluated whether aav1 - mhgck gene therapy per se may have therapeutic benefit for treating diabetes . to this end , we injected aav1 - mhgck vectors ( 10e12 vg / kg ) into the hindlimbs of c57b16 diabetic mice . experimental t1d was induced by stz administration and viral vectors ( aav1 - mhgck ) were injected 15 days after stz . a control group of stz - treated mice was injected with aav1 - null vectors ( same vector capsid but without expression of any transgene ). two - months after aav injection an insulin tolerance test was performed using low doses of insulin ( 0 , 375 u / kg ). fig1 shows that aav1 - mhgck treatment dramatically increase glucose uptake and reduce glycemia in the presence of exogenous insulin . these results indicate that gene therapy with aav1 - mhgck could be combined with regular exogenous insulin injections to improve the conventional treatment of t1d diabetes . the inventors performed experiments in high fat fed animals as a model of t2d . in these animals , aav1 - rgck vectors ( 2e12 vg / kg ) were injected in hindlimb muscles before the induction of diabetes by the high fat diet ( hfd ). three months after hfd an intraperitoneal insulin tolerance test ( 0 , 75 u / kg ) was performed . insulin sensitivity of aav1 - gck - treated mice was similar to control healthy mice while hfd fed mice were insulin resistant ( fig1 ). these data demonstrate that aav1 - gck gene therapy per se could be considered as a treatment for diabetic patients in which insulin production is still present , such as early phases of t2d patients . 1 . 1997 . report of the expert committee on the diagnosis and classification of diabetes mellitus . diabetes care 20 : 1183 - 1197 . 2 . eizirik , d . l ., and mandrup - poulsen , t . 2001 . a choice of death — the signal - 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