Patent Application: US-201715429132-A

Abstract:
the present invention concerns in general novel fusion proteins comprising a membrane transferring moiety and an enzymatic moiety . the present invention further concerns a method of treating disease using said fusion proteins .

Description:
in one embodiment , the present invention provides a composition for treating or alleviating a mitochondrial disorder , comprising a fusion protein , wherein the fusion protein comprises a protein transduction domain ( ptd ) fused to a functional component of an enzyme of a mitochondrial multi - component enzyme complex . in certain preferred embodiments , the fusion protein is produced by recombinant techniques as provided herein , provision of pto - fusion proteins containing a catalytic domain of a mitochondrial enzyme to a subject in need thereof is capable of treating and alleviating mitochondrial metabolic disorders . in certain preferred embodiments , the fusion protein of methods and compositions of the present invention further comprises a mitochondria targeting sequence ( mts ). the mts is preferably selected from the group consisting of ( a ) the naturally occurring mts of the mitochondrial enzyme or ( b ) an mts of another mitochondrial enzyme that is encoded by the nuclear dna , translated / produced in the cytoplasm , and transported into the mitochondria . in other preferred embodiments , such as those exemplified herein , the mts is that of the mitochondrial enzyme whose catalytic domain is present in the fusion protein . thus , the entire wild - type sequence of the enzyme , or a fragment thereof containing both the mts and the catalytic domain , may be used in fusion proteins of the present invention . it will be understood to those skilled in the art that the mts &# 39 ; s of various mitochondrial enzymes synthesized in from nuclear genes are largely if not completely interchangeable , and thus may be used in an interchangeable fashion in methods and compositions of the present invention . in certain preferred embodiments , the mts is situated between the pto and the enzyme or functional component thereof , as the case may be . in certain more preferred embodiments , the portion of the fusion protein c - terminal to the mts consists of the functional component of an enzyme . in another embodiment , no residues heterologous to the enzyme are present c - terminal to the mts . in this embodiment , cleavage of the mts generates an enzyme with the native sequence , thus able to readily integrate into a conformationally - sensitive multi - component enzyme complex . in certain preferred embodiments , the ptd is a tat peptide . in other embodiments , the ptd is another ptd known in the art that is capable of traversing the cellular and mitochondrial membranes of a eukaryotic cell . non - limiting representative examples of suitable ptd sequences are listed herein . each type of fusion protein represents a separate embodiment of the present invention . in another embodiment , the present invention provides a pharmaceutical composition for treating or alleviating a mitochondrial disorder , comprising a pharmaceutically acceptable carrier and as an active ingredient a fusion protein of the present invention . in another embodiment , the present invention provides use of a fusion protein of the present invention for the preparation of a medicament for the treatment of a mitochondrial disorder . in another embodiment , the present invention provides a method for treating a mitochondrial disorder , the method comprising the step of administering to a subject in need of such treatment a therapeutically effective amount of a fusion protein of the present invention , thereby treating a mitochondria disorder . upon entry into a mitochondrion of the subject , the fusion protein restores the missing enzymatic activity . in another embodiment , the present invention provides a method for introducing a mitochondrial enzyme activity into a mitochondria of a subject , the method comprising the step of administering to a subject in need of such treatment a . therapeutically effective amount of a fusion protein of the present invention , thereby introducing a mitochondrial enzyme activity into a mitochondria of a subject in need thereof as provided herein m examples 1 - 4 , tat - lad is able to enter cells and their mitochondria rapidly and efficiently . moreover , it is able to raise lad activity within lad - deficient cells and their mitochondria back to normal activity values and higher . most importantly , it is able to replace the mutated enzyme and be naturally incorporated into α - ketoacid dehydrogenase complexes such as the pdhc . we sow here that pdhc activity of lad deficient cells treated with tat - lad changed from ˜ 10 % to 70 - 75 % of normal activity after only 3 hr ′ of incubation . these high enzymatic activity values decreased following 24 hr ′ of incubation but stably remained well above basal activity . thus , in a clinical context , a single application may be sufficient for a patient presenting with a life - threatening decompensation episode . one advantage of using tat - fusion proteins for treatment of mitochondrial disorders is their ability to be delivered into virtually all cells with no specificity . when trying to replace a mutated mitochondrial enzyme there is no need for specific targeting but rather to deliver the enzyme into each cell / tissue , reaching primarily high - energy demanding tissues such as muscles , liver , and central nervous system ( cns ), which are usually the most affected in these types of disorders . moreover , lad - tat exhibited a very rapid mode of action , raising whole - cell lad activity in lad - deficient cells back to normal values after only 30 min incubation and even higher values upon prolongation of treatment ( fig2 d - e ). normal lad activity in fibroblasts ranges between 60 - 140 nmol / min / mg and in asymptomatic carriers of lad deficiency between 25 - 50 nmo / min / mg ( berger , 1996 ). the pdhc is a macromolecular multi - component enzymatic machine . its assembly process involves numerous different subunits . optimal positioning of individual components within this multi - subunit complex directly affects the efficiency of the overall enzymatic reaction and the stability of its intermediates ( vettakkorumakankav , 1996 ; berger , 1996 ; del gaizo 2003b ). given the structure of the complex , restoration of activity of a whole complex reduced due to a single mutated nonfunctioning component would not have been expected to be treatable by exogenous administration of the mutated component . interestingly , as demonstrated herein , tat - mediated replacement of the e3 component was sufficient to increase the enzymatic activity of the whole complex of the pohc ( fig5 ). as provided herein , ptd fusion proteins of the present invention raised pohc activity four - to fivefold in a sustained fashion , through the last timepoint at 24 hours ( fig5 b ). when treating a metabolic disease such as lad deficiency , there is no need to augment enzyme activity back to 100 %; rather , it need by raised above the energetic threshold required for a normal metabolism . even slight augmentation in lad activity can raise atp synthesis rate and can favorably affect the neurological involvement in lad deficiency . therefore the changes demonstrated herein in lad activity , lad / cs ratio , and pohc activity are likely to significantly affect clinical presentation in patients at least to the level of asymptomatic lad deficiency carriers . today , one major impediment of ert is the inability of the administered enzyme to cross the blood - brain barrier ( bbb ). this fundamental obstacle has severely limited development of ert for metabolic disorders in which the cns is affected ( brady , 2004 ). tat - fusion proteins are able to cross bbb , thus making them a favorable choice for development of ert for metabolic disorders involving the cns . as provided herein in examples 5 - 7 , the lad deficiency of e3 mice is treatable by pto - lad proteins of the present invention . it is noteworthy that experiments with the e3 mice have established substantial evidence that alternations in α - ketoacid dehydrogenases ( the complexes containing lad ) may play a role in the pathogenesis of neurodegenerative diseases . decreases in activity of the lad - associated complexes α - ketoglutarate dehydrogenase and pyruvate dehydrogenase , in brain , represent a common element in several age - associated neurodegenerative diseases , including alzheimer &# 39 ; s and parkinson &# 39 ; s diseases ( gibson et al ., 2000 and sullivan and brown , 2005 ). studies of adult lad - deficient mice have suggested that a partial decrease of lad , which is sufficient to diminish activity of its associated enzyme complexes ( johnson et al ., 1997 ), results in an elevated level of susceptibility to chemical neurotoxicity ( klivenyi et al ., 2004 ). moreover , variations in the dld gene ( the mouse analogue of lad ) have been linked to alzheimer &# 39 ; s disease ( brown et al , 2004 and brown et al 2007 ). furthermore , ptd - lad fusion proteins of the present invention are shown herein to restore lad and pdhc activity to brain , thus showing that they can cross the bbb and functionally integrate into pdhc there . these results clearly show that ptd - lad fusion proteins of the present invention are capable of treating neurodegenerative diseases . fibroblast primary culture cells of patients bearing the mutated genotypes g229cn35x , e375k / e357k and d479v / d479v were established from forearm skin biopsies . cells were maintained in dmem ( biological industries , beit - haemek , israel ) supplemented with 15 % fetal bovine serum ( hyclone , logan ut , usa ), penicillin / streptomycin and l - glutamine ( biological industries , beit - haemek , israel ) in a humified atmosphere with 5 % co2 at 37 ° c . all cell cultures tested negative for mycoplasma contamination . all experiments involving patients &# 39 ; cells were approved by the hadassah university hospital ethical review committee . tat fusion proteins were generated using the ptat plasmid , provided by dr . s . f . dowdy . the plasmid contains a gene encoding a 6 - histidine his - tag , followed by the tat peptide ( aa 47 - 57 ). to construct a ptat plasmid with lad fused to the his - tagged tat peptide , the gene for human lad precursor was amplified by pcr from a placental cdna library using the oligonucleotides set forth in seq id no : 1 ( forward ) and seq id no : 2 ( reverse ). the pcr product was cloned downstream of the tat sequence into a bamhi / xhoi - digested ptat vector . the tat - a - lad expression plasmid was constructed by pcr amplification of the mature lad sequence from the tat - lad plasmid using the oligonucleotides set forth in seq id no : 5 ( forward ) and seq id no : 6 ( reverse ). the pcr product was cloned downstream of the tat sequence into a bamhi / xhoicut ptat plasmid . a control lad protein lacking the tat peptide was also cloned . the lad expression vector was generated by subcloning the lad fragment into a modified ptat vector lacking the tat sequence ; nucleotide and amino acid sequences of the control lad protein are set forth in ( seq id no : 45 - 46 , respectively ). all clones were confirmed by sequencing analysis . examples of the sequences used are given below : the tat - lad dna sequence —( includes his tag , tat peptide , and the gene for human lad precursor ) is set forth in seq id no : 3 . the amino acid sequence is set forth in seq id no : 4 . the naturally - occurring lad mts has the sequence set forth in seq id no : 39 . the sequence used in tat - lad is identical except that it lacks the n - terminal met and is set forth in seq id no : 41 . e . coli bl21 - codonplus ( ade3 ) competent cells transformed with plasmids encoding the fusion proteins were grown at 37 ° c . in slb medium containing kanamycin ( 50 μg / ml ), tetracycline ( 12 . 5 μg / ml ) and chloramphenicol ( 34 μg / ml ). at an od 600 of 0 . 8 , protein expression was induced by adding iptg ( 1 mm , final concentration ). after a 24 - hr incubation at 22 ° c ., cells were harvested by centrifugation ( 2000 × g for 15 min at 4 ° c .) followed by sonication in binding buffer ( pbs ph7 . 4 , pmsf 1 mm and10 mm imidazole ( sigma - aldrich , st . louis , usa )). the suspensions were clarified by centrifugation ( 35 , 000 × g for 30 min at 4 ° c . ), and the supematants containing the fusion proteins were purified under native conditions using hitraptm chelating hp columns ( amersham - pharmacia biotech , uppsala , sweden ) pre - equilibrated with binding buffer . columns were washed by stepwise addition of increasing imidazole concentrations . finally , target proteins were eluted with elution buffer ( pbs ph7 . 4 and 500 mm imidazole ). all purification procedures were carried out using the akta ™ fplc system ( amersham - pharmacia biotech , uppsala , sweden ). removal of imidazole was performed by dialysis against pbs ( ph 7 . 4 ). proteins were kept frozen in aliquots at − 20 ° c . until use . proteins ( 5 - 20 μg protein / lane ) were resolved on 12 % sos - page gels and transferred onto an immobilon - ptm transfer membrane ( millipore , bradford , usa ). western blots were performed using anti - lad ( elpeleg 1997 ), anti - his ( amersham - pharmacia biotech , uppsala , sweden ), anti - α - tubulin ( serotec , oxford , uk ) and anti - vdac ( porin ) ( calbiochem , darmstadt , germany ) antibodies at 1 : 1000 , 1 : 10 , 000 , 1 : 10 , 000 , or 1 : 5000 dilutions , respectively . cells were plated on 6 - well plates or in 250 ml flasks ( nunc brand products , roskilde , denmark ). when cells reached 90 % confluency , medium was replaced with fresh medium containing 0 . 05 - 0 . 1 mg / ml ( final concentration ) tat - fusion proteins for various time periods . after incubation , cells were washed with pbs , trypsinized , pelleted and kept at − 80 ° c . till further use . pellets were then resuspended in pbs containing 0 . 5 % triton x - 100 and 1 mm pmsf ( sigma - aldrich , st . louis , usa ), kept on ice for 10 minutes and centrifuged at 15 , 000 × g for 10 minutes . the supernatants were analyzed by western blotting analysis or for enzyme activity . mitochondrial fractions were isolated from cultured cells using a differential centrifugation technique ( bourgeron 1992 ). cells were washed with pbs , tripsinized and pelleted . the cells &# 39 ; pellets were kept frozen at − 80 ° c . till use . pellets were resuspended in ice - cold tris - hcl buffer ( 10 mm , ph7 . 6 , 1 mm pmsf ) and homogenized with a dounce homogenizer ( teflon - glass ). the homogenates were combined with sucrose ( 0 . 25 m , final concentration ) and centrifuged for 10 min at 600 × g at 4 ° c . the supernatants were collected and centrifuged for 10 min at 14 , 000 × g at 4 ° c . the resulting pellets containing the mitochondria were resuspended in pbs containing 0 . 5 % triton x - 100 and 1 mm pmsf and incubated on ice for 15 min 20 before being analyzed for enzymatic activities and western blots . purity of sub - cellular fractions was confirmed by western blotting using the following specific marker antibodies : α - tubulin for cytoplasm and vdac ( porin ) for mitochondria . lad and cs activities were determined for whole - cell protein extracts , sub - cellularfractions or purified tat - fusion proteins . lad activity was determined as described m berger , 2005 . the reaction was performed in potassium phosphate buffer ( 50 mmol / l , ph 6 . 5 ) containing edta ( 1 mmol / l ) and nadh ( 1 . 5 mmol / l ) ( sigma - aldrich , st . louis , usa ). following addition of lipoamide ( 2 mmol / l ) ( sigma - aldrich , st . louis , usa ), the decrease in absorbance from a steady state was measured spectrophotometrically at 340 nm ( uvikon xl , bio - tek instruments , milan , italy ). cs activity was determined by following spectrophotometrically ( 412 nm ) the appearance of free sh - group of the released coa - sh upon the addition of 10 mm oxaloacetate to sub - cellular fractions to which 100 um acetyl - coa and 2 mm dtnb ( dithionitrobenzoic acid ; sigma - aldrich , st . louis , usa ) was added . analysis of cells treated with tat - lad by fluorescence and confocal microscopy tat - lad and lad proteins were fluorescently labeled with fluorescin ( fitc ) using a protein labeling kit ( ez - label , pierce biotechnology , rockford ill ., usa ) according to the manufacturer &# 39 ; s protocol . unbound fluorescent dye was removed by dialysis against pbs . cells grown on coverslips to 50 - 70 % confluency were treated with fitc - labeled tat - lad or lad ( 0 . 1 mg / ml final concentration ) for various time periods . when indicated , cells were further incubated with the mitochondrial selective fluorescent dye mitotracker - red cmxrostm ( molecular probes , eugene , usa , 200 nm ). cells were then washed with pbs , fixed in 3 . 7 % formaldehyde in pbs for 10 min at room temperature , and washed again . in fluorescence experiments , cells were analyzed directly without fixation . cells were analyzed with a fluorescence microscope . ( nikon 90 nikon corporation , tokyo , japan ) or a confocal laser scanning microscope ( nikon cl , nikon corporation , tokyo , japan ). pdhc activity was determined using radioactive pyruvate as follows : frozen cell pellets were suspended and sonicated in 0 . 25 ml potassium - phosphate buffer ( 10 mm , ph 7 . 4 ). the reaction was performed in 0 . 4 ml reaction buffer containing 200 - 300 μg protein whole - cell extracts and was terminated by adding 1m perchloric acid . the 14 co 2 was collected in hyamine hydroxide ™ ( packard , usa ) and counted in a liquid scintillation ( ultimagold ™, packard , usa ) counter ( kontron instruments , zurich , switzerland ). controls with no coenzymes were conducted simultaneously to account for background 14 co 2 release . mitochondria isolated from healthy fibroblasts and radioactive - labeled tat - lad protein and control tat - lad protein were used . in vitro translation of the proteins was performed using the tnt quick coupled transcription / translation system ™ ( promega , madison , wis .) in the presence of [ 35 s ]- methionine ( amersham biosciences , piscataway , n . j .). isolated mitochondria were incubated with the radio - labeled proteins ( 1 mg / ml mitochondria , 1 : 10 volume - to - volume ratio ) for 30 minutes at 30 ° c ., then pelleted , washed with buffer a , and treated with 2 . 5 μg / ml proteinase k ( roche diagnostics , mannheim , germany ) for 10 minutes on ice . phenylmethylsulphonylfluoride was added ( 1 mmol , final concentration ) to stop the reaction . mitochondria were then re - pelleted , washed , and analyzed using 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis gels that were fixed , dried and visualized using a phosphorimager ™ ( bas - 2500 ; fujifilm , valhalla , n . y .). over - expression and purification of the fusion protein tat - lad was accomplished by inserting the precursor human lad sequence into the ptat vector . expression vectors encoding tat -. 1 - lad , lacking the mts sequence , and a control lad protein lacking the tat peptide were also constructed ( fig1 a ). these proteins were all expressed and highly purified under the same conditions . sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and western blotting confirmed the identity of these highly purified proteins ( fig1 b ). these purified lad - based fusion proteins were found to be highly active in an in vitro lad enzymatic activity assay ( fig1 c ). the next experiment examined the ability of protein transduction domains ( ptd &# 39 ; s ) such as tat to deliver the human lad enzyme into cultured cells from patients with lad deficiency . purified tat - lad was incubated for different time periods with cells from patients heterozygous for the g229c / y35x and e375k lad mutations . whole - cell protein extracts were prepared and analyzed by western blotting using anti - lad antibodies . tat - lad fusion protein ( 58 kda ) rapidly entered g229c / y35x cells and was detectable after 30 minutes of incubation ( fig2 a ). in cells homozygous for the e375k mutation ( fig2 b ), its delivery was somewhat slower ; it was detected within the cells after a 2 - hour incubation . endogenous mutated lad ( 50 kda ) was detected only in g229c / y35x cells and not in e375k cells ( fig2 a ). in both cell lines , steady state was reached after 2 - 3 hours ; thus , the amount of the fusion protein remained similar through the 6 - hour ( fig2 a ) and 24 - hour ( fig2 b ) timepoints . delivery of tat - lad into cells was also followed using direct fluorescence analysis . tat - lad was fluorescently labeled with fluorescin ( fitc ), incubated with g229cn35x cells for different time periods , and analyzed by fluorescence microscopy . fitc - labeled lad protein lacking the ptd moiety was used as a control protein . tat - lad was efficiently delivered into the cells ( fig2 c , panels 1 - 3 ) whereas fluorescence signals were not detected in cells treated with the control lad protein ( fig2 c , panel 4 ). these results correlated with the western blot analysis ( fig2 a - b ). tat - lad was detected rapidly within cells ( after only 30 . min of incubation ; fig2 c , panel 1 ) and there were no differences in fluorescence signal intensity after longer incubation periods ( fig2 c , panels 2 - 3 ). to test the ability of a ptd to deliver an active human lad enzyme into lad - deficient cells , purified tat - lad was incubated with g229cn35x and e375k cells for different time periods . these experiments utilized the control lad protein and tat - pah protein , which is a control tat - fusion enzyme that lacks lad activity . protein extracts of treated cells were analyzed for their lad activity . activity of lad within the cells increased dramatically in concordance with incubation time , reaching steady state after 2 - 3 hr ′ ( fig2 d - e ). these results resembled those observed by the western analysis . this augmentation in lad activity within patients &# 39 ; cells was dose - dependent , and was not observed following addition of control lad protein . in g229cn35x cells , lad activity increased by 2 . 5 - fold ( from 31 nmol / min / mg to 78 nmol / min / mg ) after only 30 min of incubation and reached equilibrium of 230 - 250 nmol / l min / mg , an 8 - fold increase , after 2 - 3 hr ′ ( fig2 d ). g229cn35x cells incubated with control proteins tat - pah or lad showed no change in basal lad activity , & gt ; 20 nmol / min / mg , which is lower than normal values ( saada 2000 ). in e375k cells ( fig2 e ), the same trends were observed . lad activity increased by − 9 fold ( increasing from 5 nmol / min / mg to 423 nmol / min / mg ) after 2 hr ′ of incubation and reached equilibrium of 630 - 690 nmol / min / mg after 4 hr ′ of incubation , which lasted through the last time 24 hr ′ incubation . e375k cells that were incubated with the control protein lad showed no change in their basal lad activity . though treated with identical protein concentrations , e375k and g229cn35x cells responded differently as maximum activity values were much higher in e375k than m g229cn35x cells , indicating possible differences in treatment efficiency in patients bearing different genotypes . the next step was to examine the ability of tat - lad to be delivered across the mitochondrial membrane and naturally processed in mitochondria . in vitro - translated [ 35 s ]- methionine - labeled tat - lad was incubated with isolated mitochondria from healthy fibroblasts . the mitochondria were treated with proteinase k to digest proteins nonspecifically adsorbed to the outer membrane , thereby ensuring that the mitochondrial extract contained only proteins within the mitochondria . as a control , 35 s - methionine - labeled tat - lad protein lacking the mts ( and consequently lacking the natural processing site within it ) was used . as seen in fig3 a , tat - lad and tat - δ - lad were both expressed at their expected molecular sizes of 58 and 54 kd , respectively . after treatment , both tat - lad and tat - δ - lad were detected within the mitochondria after 30 minutes of incubation ( fig3 b ), because of the ptd sequence that these proteins carry . however , only the tat - lad fusion protein was processed to its mature size , as indicated by the appearance of an additional 50 - kd band on the sos - page autoradiograph ( fig3 b , asterisk ). as expected , the tat - δ - lad protein ( lacking the mts ) was not processed , and appeared as a single band at its full unprocessed size . thus , tat - lad is able to be delivered into mitochondria and processed therein . it was next examined whether tat - lad was able to reach mitochondria after being delivered into intact cells . purified tat - lad was incubated with g229cn35x and d479v cells for different time periods . after incubation , mitochondrial and cytoplasmic sub - cellular fractions were prepared and analyzed for presence of tat - lad and for lad enzymatic activity . cs activity was utilized as a mitochondrial marker . western blot of sub - cellular fractions indicated the presence of tat - lad ( 58kda ) in both cytosolic and mitochondrial fractions of treated g229cn35x and d479v cells following 4 and 6 hr ′ of incubation ( fig4 d and h , respectively ). purity of sub - cellular fractions was confirmed using antibodies against the sub - cellular markers α - tubulin ( 50 kda ) for the cytoplasm and vdac ( porin ) ( 31 kda ) for the mitochondria . in support of these findings , there was a significant increase in lad activity in both cytosolic and mitochondrial fractions of cells treated with tat - lad . in g229cn35x cells , lad activity in mitochondrial fractions increased by 7 - fold ( from 28 nmolmin / mg to 205 nmolmin / mg ) after a 4 hr ′ incubation ( fig4 a ). enzymatic activity remained about the same after 6 hr ′ ( 193 nmolmin / mg ) demonstrating that equilibrium had been reached . this dramatic increase in lad activity was also measured in cytosolic fractions , changing from 10 nmomin / mg to 222 and 339 nmolmin / mg after 4 hr ′- and 6 hr ′ incubations , respectively ( fig4 a ). similar results were observed with d479v cells . lad activity in mitochondrial fractions changed from 28 nmolmin / mg to 165 and 117 nmolmin / mg after 4 hr ′- and 6 hr ′ incubations , respectively ( fig4 e ). in cytosolic fractions , activity changed from 20 nmolmin / mg to 125 and 193 nmolmin / mg after 4 hr ′ - and 6 hr ′ incubations , respectively . in addition , cs enzymatic activity was determined in g229cn35x cells ( fig4 b ) and d479v cells ( fig4 f ). cs is a mitochondrial matrix enzyme that participates in the krebs cycle , converting acetyl - coa to citrate . cs enzymatic activity assay was used as a control reference to verify the purity of mitochondrial sub - fractions and also to calculate lad / cs ratio to standardize lad enzymatic activity values . in both cell lines , cs activity in cytosolic fractions was barely detectable , while in the mitochondrial fractions it was within the range of normal levels for fibroblasts , thus verifying the purity of sub - cellular fractions . furthermore , cs activity was constant and almost identical in all mitochondrial fractions , enabling proper standardization of lad activity values . mitochondria of g225c / y35x exhibited lad / cs ratios of 0 . 102 before incubation and 0 . 740 and 0 . 678 after 4 hr ′- and 6 hr ′ incubations with tat - lad , respectively ( fig4 c ). in mitochondria of d479v treated cells , the lad / cs ratio changed from 0 . 142 to 0 . 715 and 0 . 561 after 4 hr ′- and 6 hr ′ incubation , respectively ( fig3 g ). co - localization experiments were used to further confirm delivery of tat - lad into the mitochondria of lad - deficient cells . fitc - labeled tat - lad was incubated with g229cn35x cells grown on coverslips for different time periods . cells were then incubated with the mitochondrial - selective fluorescent dye mitotracker - red cmxrostm and analyzed by confocal microscopy . as shown in fig5 a , tat - lad ( green fluorescence , middle column ) co - localized with mitochondria ( red fluorescence , left column ) within the first 30 minutes of incubation , as indicated by the yellow staining in the merge ( right column ). the final and most crucial test for tat - lad &# 39 ; s ability to successfully treat lad deficiency by ert is the enzyme &# 39 ; s ability to substitute for the mutated endogenous enzyme , including successful integration into its natural multi - component enzymatic complexes such as pyruvate dehydrogenase complex ( pdhc ). lad deficiency affects three mitochondrial multi - component enzymatic complexes , whose activity could be restored by tat - lad . the ability of tat - lad to successfully replace the endogenous defective enzyme and increase the activity of pdhc was tested in d479v and e375k cells . pdhc activity was increased in the two genotypically different cells . in e375k cells , pdhc activity increased significantly by 12 - fold after 3 hours of incubation ( from 0 . 029 to 0 . 367 nmol / min / mg ), remaining approximately four - to fivefold higher than the low basal values for at least 24 hours ( fig5 b ). presented as a percentage of normal pdhc activity of healthy fibroblasts , the pdhc activity in d479v cells increased from 9 % to 69 % of normal activity after 3 hours of incubation , remaining at 50 % of the normal level for at least 24 hours . in e375k cells , pdhc activity increased from 5 to 75 % of normal activity after 3 hours incubation , declining to about 30 % after 24 hours of incubation ( fig5 c ). of note , these pdhc activity values are in close correlation with lad enzymatic activity values measured in mitochondria of treated cells , reaching maximum levels after 3 hr ′ incubation with tat - lad . ptd - lad fusion proteins are thus able to treat lad deficiency by augmenting pdhc activity in lad - deficient cells . enzymatic activity of lad in plasma of e3 mice injected with tat - lad the mouse model of lad deficiency is described in klivenyi , p . et al ( mice deficient in dihydrolipoamide dehydrogenase show increased vulnerability to mptp , malonate and 3 - nitropropionic acid neurotoxicity . j neurochem 88 : 1352 - 1360 , 2004 ) and johnson , m t et al ( targeted disruption of the murine dihydrolipoamide dehydrogenase gene ( dld ) results in perigastrulation lethality . proc natl acad sci usa 94 : 14512 - 14517 , 1997 ). these mice are heterozygotes to a recessive loss - of - function mutation affecting lad gene ( did , in mice ) expression at the mrna level ( instability ) ( did +/− mice or e3 mice ). homozygous mice die in - utero at a very early gastrulation stage . these mice are phenotypically normal , though their lad activity is reduced by ˜ 50 %, affecting all the lad - dependent enzyme complexes . similarly , humans heterozygous for lad deficiency exhibit − 50 % lad activity , but usually have no clinical symptoms . these mice are currently used in experiments in the field of neurodegenerative disorders including alzheimer &# 39 ; s , parkinson &# 39 ; s and huntington &# 39 ; s disease . a single dose ( 0 . 2 mg per mouse ) of highly purified tat - lad was injected into the tail vein of e3 mice , and several tissues were extracted and analyzed for lad and pdhc activities at different time points . several mice were used at each time point . to test the ability of tat - lad to treat lad deficiency in vivo , purified tat - lad was injected intravenously into e3 mice , and its effect on lad and pdhc activities was measured in several tissues . this experiment concentrated our on 3 major organs that have the highest energy demands and thus are often affected in mitochondrial disorders — the liver , the heart ( muscles ) and the brain . first , behavior and stability of the injected fusion protein tat - lad in the plasma of injected mice were characterized by measuring lad enzymatic activity . blood samples from e3 injected mice were withdrawn at different time points , and plasma was prepared . no lad activity is present in the plasma of either normal healthy mice or e3 mice , so the lad activity at the first time point was set as the reference . following the first time point , a decrease in lad activity was observed in the plasma of e3 mice , over time ( fig6 ). to determine whether a component or factor exists in the plasma that reduced lad activity over time ; mouse plasma was incubated with tat - lad in vitro under the same concentrations : at 37 ° c . and for the same time periods . lad activity remained stable in these plasma samples . thus , the decrease in lad enzymatic activity in the plasma was a result of delivery of tat - lad into the organs and tissues of mice . indeed , these results correlate with the lad activity measured within the organs ( fig7 below ). the lad control protein , lacking the tat delivery moiety , also decreased its activity in plasma overtime , suggesting possible clearance mechanisms in this case . organs were harvested from the mice described in the previous example , and lad activity there was measured . fig7 a depicts the percentage increase from the basal activity measured in the heterozygous mice , namely e3 , non - treated mice , injected only with pbs . a single intravenous injection of tat - lad ( 0 . 2 mg per mouse ) significantly increased lad enzymatic activity within the liver , heart and most importantly — in the brain after only 30 minutes . the shapes of the curves were similar in the brain and heart and slightly different in the liver ( fig7 c - d and b , respectively ). even more robust increases were observed at steady state . in liver , lad activity reached a steady state at about 40 % of non - treated mice and remained at the same level for up to 6 hours , while in brain and heart , steady - state lad activity was higher , peaking at 4 hours at levels of 80 % and 100 %, respectively . the lad control protein , lacking the tat delivery moiety , injected in the same amount and under identical conditions , did not significantly increase in lad activity in the organs . in addition and also of importance was the fact that 24 hours following the injection , lad activity was still 10 % higher than the basal activity . thus , pto - lad fusion proteins are able to fully restore deficient lad activity in a lad - deficient disease model and thus are able to treat acute decompensation episodes . the long - term magnitude of the increase after only a single treatment , 10 %, is also sufficient to affect the clinical status of many cases . principle of pdhc activity measurement in mice &# 39 ; s tissues . a kit from mitosciencestm ( catalog no . msp18 ) for measuring pdhc enzymatic activity was used . pdhc was immuno - captured from tissue lysates , and its enzymatic activity is measured . this ensured that any increase in the measured pdhc activity resulted only from the tat - lad that had become integrated into the pdhc complex . the enzymatic assay measures reduction in nad + to nadh by an increase in absorbance at 340 nm . the next experiment directly tested the ability of ptd - lad fusion proteins to substitute for the mutated endogenous enzyme , following successful integration into its natural multi - component enzymatic complexes , in the organs of the tat - lad - injected mice described in example 5 . fig8 a depicts the percentage increase over basal pdhc activity of untreated e3 mice ( mock - treated by injection with pbs ) in each organ . brains and hearts ( fig8 c - d , respectively ) of treated e3 mice both responded robustly to tat - lad treatment ; peaking at 4 hours , with a 145 % increase in pdhc enzymatic activity ; liver samples ( fig8 b ) peaked at 2 hours with a 135 % increase in the activity . a substantial and significant increase in pdhc enzymatic activity ( 40 - 65 %) was also evident in the three organs at 24 hours post - treatment . treatment with the control protein lad did not affect the basal pdhc activity . interestingly , the percent increase in pdhc activity was much greater than that of lad activity in the tissues , highlighting the potency of the fusion proteins used ( fig9 a - c ). thus , a single application of a ptd - lad fusion protein is able to significantly increase pdhc activity in a disease model of lad deficiency . pto - lad fusion proteins are thus able to treat and ameliorate lad deficiency pathologies . tat - orf66 restores complex i activity in the cells of a patient with nadh : ubiquinone oxidoreductase ( complex n deficiency in order to construct a plasmid expressing a tat - c60rf66 fusion , the gene for human c60rf66 was amplified by pcr from lymphocytes complementary dna library , using the oligonucleotides set forth in seq in no : 47 ( forward ) and seq in no : 48 ( reverse ). the pcr product was cloned downstream of the tat sequence into a bamhi / xhoi - digested ptat fragment . a missense mutation in a conserved residue of the c60rf66 gene has been identified in a consanguineous family that presented with infantile mitochondrial encephalomyopathy attributed to isolated nadh : ubiquinone oxidoreductase ( complex i ) deficiency . in muscle of patients , levels of the c60rf66 protein and of fully assembled complex i were markedly reduced . transfoction of the patients &# 39 ; fibroblasts with wild - type c60rf66 cdna restored complex i activity ( saada a et al , c60rf66 is an assembly factor of mitochondrial complex i . am j hum genet 82 ( 1 ): 32 - 8 , 2008 ). the mrna sequence of c60rf66 is set forth in seq id no : 7 ( genbank accession 10 # nm_014165 ). the amino acid sequence of the product of c60rf66 is set forth in seq id no : 8 ( genbank accession # nm_014165 ). the first 34 residues of the protein , ( seq id no : 9 ), are predicted by the targetp software to form the mitochondrial - targeting sequence ( saada a et al , ibid ). to test the ability of a tat - fusion protein to treat complex i deficiency , a tat - c60rf66 fusion protein was constructed and highly purified . primary fibroblast cells isolated from a patient with the missense mutation in the c60rf66 gene were incubated with tat - orf66 for 48 hr , and mitochondria were isolated and analyzed for complex i activity . the tat - fusion protein was able to restore 80 % of wild - type complex i activity in the mitochondria ( fig1 ). the findings presented herein demonstrate that a variety of mitochondrial enzymes can be successfully treated by ert using ptd - based fusion proteins . deficiencies in lad , an enzyme that forms part of several multi - component enzymatic complexes , and c60rf66 , an assembly factor of complex i , were successfully treated . of note , the enzymes were able to translocate into the mitochondria and function in the conformation - sensitive context of these enzymatic complexes with their activity intact , following removal of the heterologous parts of the molecule . the findings presented herein demonstrate that a variety of mitochondrial metabolic disorders are treatable by ert using ptd - based fusion proteins , as evidenced by treatment of both lad deficiency and complex i deficiency . chinnery , p . f . & amp ; schon , e . a . mitochondria . j . neurol . neurosurg . psychiatry 74 , 1188 - 1199 ( 2003 ). brautigam , c . a ., chuang , j . l ., tomchick , d . r ., machius , m . & amp ; chuang , d . t . crystal structure of human dihydrolipoamide dehydrogenase : nad +/ nadh binding and the structural basis of disease - 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