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University of Florida | Journal of Undergraduate Research | Volume 13 Issue 3 | Summer 2012 1 Inhibition of I1PP2A as a Therapeutic Strategy for Tauopathy Matthew D Isaacson D r. Aaron C. Hirko, and Dr. Michael A. King College of Medicine, University of Florid a Tauopathies a class of neurodegenerative diseases, the most notable of which is Alzheimer's disease (AD) are characterized by the hyperphosphorylation of tau, a protein responsible for stabilizing microtubules inside cells (primarily in the CNS). I1PP2A, an endogen ous protein inhibitor of the most prominent tau phosphatase, PP2A, is known to be elevated in AD. Heightened levels of I1PP2A alone could account for the hyperphosphorylation of tau, making it a prime target for treatment. The goal of this project wa s to develop an inhibitor of I1PP2A that has potential as a therapeutic treatment a nd show that treatment with this inhibitor wil l increase PP2A activity levels. Increasing I1PP2A concentration in mouse hippocampal cell lysates (to simulate tauopathy) decr ease d the activity of PP2A by an average of 19% (p = 0.073 ), as expected Two possible inhibitors of I1PP2A its antibody and a batch of RN A aptamers selective to I1PP2A ( developed with SELEX and his tagged I1PP2A bound to Ni NTA columns ) increased PP2A activity by an average of 2 2% when treated to these lysates (p = 0.209). The se observed trends lend support to our hypothesis. The RNA aptamers generated have therapeutic potential as they may be able to be expressed insid e neurons affected by tauopathy if DNA complements were successfully packaged into r AAV vectors and transfected into the cells. I NTRODUCTION Tauopathies are a class of devastatingly progressive neurodegenerative diseases the most notable and prevalent is (AD) for which no effective treatment currently exists. The shared feature of this group of conditions is the accumulation of polymers formed by the protein, tau  specifically, two features are diagnostic: one is the formation of plaques formed by amyloid prec ursor protein (APP ) and other cellular debris, and the other is the formation of paired helical filaments (PHF) of tau contained in neurofibrillary tangles. T he plaques of APP are the subject of many research projects working toward treating tauopathy; h owever, the y are not present in all tauopathies, and the presence of abnormal tau is more closely correlated to memory difficulties and dementia ( prime sympto m s of tauopathy) [ 2 ] to stabilize microtubules through binding to tubulin, though tau can exist in various forms of phosphorylation which alters its performance Hyperphosphorylation causes tau to dissociate from tubulin, causing microtubule destabilization and PHF formation (Figure 1 ) Bundles of destabilized microtubules (neurofibrillary tangles) interfere with normal neuronal cell functions such as synaptic transmission and intrac ellular transport, and can lead to cell death [ 3 ] ( Figure 2 ) Figure 1 Microscopic view of abnormal bundles of tau.
MATTHEW D ISAACSON D R A ARON C H IRKO AND DR M ICHAEL A K ING University of Florida | Journal of Undergraduate Research | Volume 13 Issue 3 | Summer 2012 2 Figure 2 Mechanism of typical development of tauopathy. Tau is known to contain many phosphorylation sites [4 ] though several serine (ser) and threonine (thr) residues are known to be directly involved with normal tau function. Increased phosphorylation on these sites alone has shown to alter the function of tau and cause increase d PHF formation [ 5 ]. Many phosphatases exist in neuronal cells which act specifically on ser/thr residues, though the most prominent is p rotein phosphatase 2 A (PP2A) [ 6 ]. Dephosphorylating tau is not as it is also responsible for regulating signaling pathways in concurrence with other kinases and phosphatases, and even for s tabilizing microtubules directl y [ 7 ] [8 ] Decreased activity of PP2A, which is observed in AD [ 9 ], has been hypothesized to account for tau hyperphosphorylation [ 10 ], though the reason for this diminished activity is not certain. An endogenous inhibitor of PP2A, named I1PP2A (also called ANP32A or PHAP1) has been shown to be elevated in AD [ 11 ]. The increased inhibition of PP2A by I1PP2A alone could hypothetically account for diminished PP2A and tau hyperphosphorylation, though I1PP2A has also been observed to compete with tau for microtubule binding sites [ 12 ] increasing its likelihood as being a major component in tauopathies Based on these findings I1PP2A looks to be an attractiv e target for treating tauopathy, though no suitable drug candidates are known to exist. Computational drug design could be utilized to develop a therapeutic inhibitor to I1PP2A, though this method is very expensive and laborious T he use of aptamers however, has shown to be an economical, efficient and clinically safe alternative [ 13 ] Aptamers are small oligonucleotides (typically 20 100 nucleot ides in length) that are selected from a large random sequence pool for binding to a target with high affinity and specificity [ 14 ]. DNA strands synthesized through phosphoramidite chemistry can allow nucleotide s to be incorporated into a sequence at random. With this strategy 10 1 3 different combinations of sequences can easily be generated for oligonucleotide s only 30 bases in length. The recursive SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process is employed to select aptamers out of such a library on the basis of affinity to a target molecule. Each round of SELEX develops a batch of aptamers more selective to the target than the previous, and often 10 rounds of selection are sufficient to isolate the best candidate aptamers [15 ]. This method has been altered to incorporate many different selection techniques, such as capillary electrop horesis [16 ]; surface plasmon resonanc e [17 ] ; and similar to what is used i n this project, Ni NTA beads [18 ]. Numerous difficulties arise when attempting to deliver a drug to specific areas in the body, especially the brain. An effective therapeutic agent must successfully be able to cross the blood brain barrier and pass through cell membranes intact if it is delivered through the bloodstream. The use of recombinant adeno associated viral vectors (rAAV) may be a good solution to some of these problem s and can be developed from jus t about any oligonucleotide of appropriate size [ 19 ] such as the aptamer selected for I1PP2A AAV is capable of transfecting non dividing cells such as mature neuronal cells implicated in tauopathies ), and have even been used recently in clinical trials for other neurological diseases
INHIBITION OF I 1 PP 2 A AS A THERAPEUTIC S TRATEGY FOR T AUOPATHY University of Florida | Journal of Undergraduate Research | Volume 13 Issue 3 | Summer 2012 3 [ 20 ]. Although treatment with AAV currently requires invasive surgical procedures, the permanent nature of AAV transfection still makes it an attractive treatment strategy (regardless of the fact that no other treatme nt exists). HYPOTHESIS Increased levels of I1PP2A may be the cause of at least some forms of tauopathy through the inhibition of PP2A and subsequent hyperphosphorylation of tau. If other pathologi es are causing diminished PP2A activity or there are other reasons for tau hyperphosphorylation, decreasing I1PP2A level s could still compensate for this imbalance. The inhibition of I1PP2A should restore PP2A activity to normal levels in tauopathies, thus serving as an effective treatment strategy. Fi gure 3 Possible conditions of tau regulation. Green indicates normal levels, where as red and yellow indicate high and low levels, respectively. Case A: the properties of normal tau phosphorylation. Case B: tauopathy due to an increased amount of I1PP2A which subsequently causes increased inhibition of PP2A and decreas ed tau dephosphorylation. Case C: the addition of an I1PP2A inhibitor in case B decreases its levels to normal and balanc es out all other conditions. EXPERIMENTAL METHODS I1PP2A Production and Purification For this project, many assays and procedures required the use of I1PP2A, which is not economically available in large quantities. This required production of I1PP2A in our own lab. Plasmids incorporating a gene for recombinant, histidine tagged I1PP2A p roduction were ordered for this purpose (QAIgenes E. C oli Expression Kit ANP32A). The protocol included with the kit was followed for transfection of competent E. coli cells, growth of these cells, and induced expression with IPTG. Following expressio n, cell cultures were lysed with lysozyme and Benzonase nuclease and centrifuged to pellet the cellular debris, as directed by the kit. I1PP2A in the supernatant was purified using Ni NTA spin columns (QAIGEN 3014), following this the supplied reagents. Samples of each step in the purification (3 washes, 4 elutions) were saved for analysis by gel electrophoresis. To remove the I1PP2A from the high salt imidazole buffer each eluate was passed through columns filled with Sephadex G 25 and washed with RNase free, DEPC treated water 100 L s ections of eluate from these columns were saved for determination of protein concentration SDS PAGE and W estern B lots 10 L of s amples to be analyzed by electrophoresis were mixed with 9.5 L of L aemmli buffer and 0.5 L of 2 mercaptoethanol and placed in a boiling water bath for 5 minutes (to denature the protein). The samples wer e then pipetted into a precast 4 15% Tris HCl Ready Gel (Bio Rad 161 1176), which was loaded for electrophoresis set at a constant 200 V. Sample and running buffers were used as directed in the Bio Rad Application Guide. The gel was removed from the machine after the dye front reached the end of the gel. Gel staining was accompli shed using Coomassie Brilliant Blue G 250 Stain Solution (Bio Rad). The protein was fixed to the gel by soaking in a 5 0% methanol, 10% acetic acid solution for 1 hour. The gel was the transferred to the G 250 staining solution for 4 hours in an orbital shaker. Afterwards, the gel was destained with a 5% ethanol and 7.5% acetic acid solution overnight For western blot analysis, the p rotein was transferred to a PVDF membrane (pre soaked in methanol) by stacking it on gel and placing in a n electrophoresis transfer block set to 80 V for 4 hours T he PVDF membrane was then removed from the block and washed with tr ansfer buffer (0.1 %v/v Tween 20 in PBS). Non specific binding sites were blocked by placing the membrane in transfer buffer with 5% nonfat milk for 1 hour in an orbital shaker. After 2 rinses with the transfer buffer the primary antibod y ( Rabbit Anti PHAP1, Chemicon Internatio nal AB4515), diluted in transfer buffer was added to the membrane and incubated again for 1 hour. This was followed with 3 more washes with transfer buffer (for 10 minutes each), after which a secondary antibody ( A nti mouse HRP ) was added to the membrane and incubated for 1 hour. Three additional washes we re done, followed by rinsing with dH 2 0. Reagents from an ECL Detection kit (GE Healthcare RPN2132) were added to the membrane as stated in the included protocol and incubated for 5 minutes. Chemifluor escence was detected with a storm molecular imager.
MATTHEW D ISAACSON D R A ARON C H IRKO AND DR M ICHAEL A K ING University of Florida | Journal of Undergraduate Research | Volume 13 Issue 3 | Summer 2012 4 Protein Concentration Assay All protein c oncentrations were determined using a Bio Rad DC Protein Assay kit (a varia t ion of the Lowry a ssay). A protein standard curve was prepa red by diluting albumin to 5 known concentrations (0.2, 0.525, 0.85, 1.175, and 1.5 mg/mL). 5 L of each sample to be analyzed was loaded into a microtiter plate. Reagents included in the kit were After mixing and a 30 minute wait period, absorbances were read in a standard microplate reader at 750 nm. Absorbances were compared to the standard curve to determine sample protein concentration s PCR, RT PCR, and Transcription Polymerase Chain Reaction (PCR) was carried out using a Phusion High Fidelity PCR kit (New England Biolabs F 5535). Reaction mixtures (50 L) were created using the reagents included with the kit protocol. The reaction mixture was subjected to 30 rounds in a thermal cycle r at temperatures of , and for denaturation, annealing, and extension, respectively To purify dsDNA from the reaction mixture, QIAquick PCR Purification kits (QIAGEN 28104) were used. All reagents and materials needed were included in the k it and its standard protocol was followed. Reverse Transcription PCR (RT PCR) was accomplished with USB One Step RT PCR Kits (Affymetrix 78350). A 50 L reaction mixture was created following the protocol and using the reagents included in the kit (enzyme mix, nucleotide mix, buffer and RNase inhibitor), starting with up to 6 L of an RNA sample. The mixture was then placed in a thermal cycler for 40 rounds under the same conditions used for PCR QIAquick PCR purification columns were also used to purify dsDNA from these reactions. To transcribe DNA oligonucleotides to RNA, Ambion MEGAshortscipt High Yield Transcription kits were used (Applied Biosystems AM1354). Reaction mixtures were reagents, then incubated for 4 hours at 37 o C. Alcohol precipitation was used to recover the RNA from this solution also following the kits protocol SELEX and A ptamer S election To begin the aptamer selection process, a library of DNA aptamers was ordered with known seque nces, as shown in Figure 4 Forward and reverse primers were also needed for these aptamers. Figure 4 DNA aptamer sequence contains 30 random nucleotides (marked by the letter N), flanked by forward and (complement) reverse primer sequences. A T7 promoter sequence is included in 1 st half of the forward primer, making transcription of the aptamers to RNA simple. Forwa rd and reverse primers sequences are also shown. The DNA aptamer library was first transcribed into RNA. The batch was then incubated with 1 mL of a 50 % slurry of Ni NTA agarose in 30% ethanol for 30 minutes at 4 o C with mild shaking. This slurry was then loaded into a 1 mL polyethylene column (with a frit to prevent the agarose from passing through column) and washed with water to recover the flow thro ugh. This batch of aptamers had thus been screened to remove any aptamers that bind directly to the Ni NTA. After 2 of these screenings, the batch of RNA aptamers was incubated with Ni NTA and a known amount of I1PP2A and loaded into a 1 mL column. The hi stidine tag on the I1PP2A binds to the Ni NTA, preventing it from
INHIBITION OF I 1 PP 2 A AS A THERAPEUTIC S TRATEGY FOR T AUOPATHY University of Florida | Journal of Undergraduate Research | Volume 13 Issue 3 | Summer 2012 5 washing off the column, and also keeps any bound aptamers held to the column. The column was then washed with water to remove any aptamers not bound to I1PP2A. The I1PP2A on the column was t hen eluted with 250 mM imidazole which competes with the his tag for binding to nickel removing any bound aptamers from the column as well. A sample of the eluate was sent through RT PCR and purified for dsDNA, which generates a batch of DNA whose RNA complement is more selective to I1PP2A than the previous batch. This process was repeated 10 times to isolate aptamers with very high affinity to I1PP2A, as shown in Figure 5 Figure 5 Process of aptamer selection used in this project. In the first 4 rounds of this process, 10 g of I1PP2A was incubated with Ni NTA and the batch of aptamers To increase the stringency of aptamer selection this number was reduced to 6 g in rounds 5 7 and 2 g in rounds 8 10. PP2A Activity Assay PP2A activ EnzChek Serine/Threonine Phosphatase Assay Kit (Molecular Probes R 33700). To prepare a fluorescent standard curve, 100 L of reaction buffer (included with the kit) was added to each well of the reference standard s trip of the microplate, then mixed to solubilize the substrate in the wells. Samples to be analyzed were diluted to 100 L with reaction buffer and added to the microplate (also with mixing). Negative controls were created by adding only reaction buffer t o blank wells. The microplate was then incubated at room temperature (guarded from light) for 30 minutes, after which fluorescence was read with a fluorescence microplate reader, set to excitation and emission wavelengths of 355 and 460 nm, respectively. T he fluorescence related directly to the amount of PP2A activity in the sample. R ESULTS The SDS PAGE gel, shown in Figure 6 shows bands at approximately 28 and 56 kD. I1PP2A is known to be 28 kD in size, so these bands could be monomers and dimers of I1PP2A, respectively No bands are visible in wash 3, showing that only 2 washes we re necessary. Four elutions were completed in total, though the 3 rd and 4 th had no visible bands and are not shown. Nearly all of the I1PP2A appeared to wash off the column in the 2 nd elution.
MATTHEW D ISAACSON D R A ARON C H IRKO AND DR M ICHAEL A K ING University of Florida | Journal of Undergraduate Research | Volume 13 Issue 3 | Summer 2012 6 Figure 6 SDS PAGE gel of the purification of I1PP2A from E. coli lysates Lane 1: protein ladder of known sizes L ane 2: cell lysate s L ane 3 flow through of the lysate passed through the Ni NTA column L ane 4: sample of 1 st wash with 10 mM imidazole L ane s 5 and 6 : 2 nd and 3 rd wash es with 20 mM imidazole L ane s 7 and 8 : 1 st and 2 nd el uate with 250 mM imidazole A western blot of the 2 nd elution shown in Figure 7 confirms the identity of I1PP2A. Lane 2 only shows 1 significant dark band at approximately 56 kD, likely indicating an I1PP2A dimer. Lanes 3 and 4 have darkest bands at 28 kD and 56 kD, as expected. Many other li ghter bands show ed immunoreactivity with the primary antibody (specific for I1PP2A) indicating that the majority of the bands in the 2 nd elution of Figure 6 are indeed fragments or complexes of I1PP2A, signifying high purity of the desired protein. Figure 7 The western blot of a sample from the 2 nd elution of pu rified I1PP2A (shown in Figure 6 ), run on an SDS PAGE gel Lane 1 : the standard protein ladder. Lane 2 : a sample of I1PP2A which was not boiled and no 2 mercaptoethanol was added prior to being run on the electrophoresis gel. Lane 3 : a sample with mercaptoethanol added but was not boiled. Lane 4 : a sample with mercaptoethanol added and boiled for 5 minutes. The results of the PP2A dephosphorylative activity assay are shown in Figure 8. The addition of extra I1PP2A significantly decreased the activity of PP2A in all cases except for the 1 st set. The addition of PHAP1 antibody increased PP2A activity, and the addition of the final batch of apta mers in creased it even further. Figure 8 D isplays the amount of fluorescence detected for each sample labeled. Each column shows the average and standard deviat ion of 3 duplicate measurements The red columns contained only cell lysates, incubated with or without 1 g of added I1PP2A. The green columns show lysates incubated with 1ug of an I1PP2A antibody with or without I1PP2A and the blue column s show lysates incubated with the aptamer batch ( amount not known) after the 10 th round of SELEX with or without added I1PP2A D ISCUSSION AND CONCLUSIONS As shown in Figures 6 and 7 I1PP2A was successfully produced by transfection of E. coli cells, retained it s
INHIBITION OF I 1 PP 2 A AS A THERAPEUTIC S TRATEGY FOR T AUOPATHY University of Florida | Journal of Undergraduate Research | Volume 13 Issue 3 | Summer 2012 7 activity, and was recovered with good purity. Figure 7 shows that I1PP2A unfolds easily with 2 mercaptoethanol without any need for boiling. An interesting observation is that w ithout 2 mercaptoethanol it seems to exist almost exclusively in the dimer form. I1PP2A contains only 2 thiol groups in its entire structure, leading to the possible conclusion that a disulfide bond exists between molecules. Since no larger complexes seem to forms, it appears that I1PP2A contains only one o f these thiol groups exposed to the environment, free to react with other thiol groups, while the other is well hidden inside the folded protein. As shown in Figure 8 PP2A a ctivity was decreased 19% on average upon the addition of extra I1PP2A (p = 0.073). The activity of PP2A somehow increase d after the addition of I1PP2A in the pl ain cell lysate sample (2 nd red column) This may have been caused by monomeric I1PP2A becoming inactivated by dimerization with large amounts of added I1PP2A. Another reason could have simply been human error, though the experiment should be repeated in either case The activity also appeared to increase in the presence of an antibody to I1PP2A, and more so by the addition of aptamers selected for I1PP2A (p = 0.209). The visible trend does lend support to our hypothesis and it is encouraging that the apt amers may inhibit I1PP2A more successfully than the antibody However, the amount of aptamers added to the samples were not known, so the large increase in activity in the last set of columns could have been due to the addition of a very large amount of mo derately effective aptamers. It appears that neither the antibody nor the aptamers were at all successful in counteracting the addition of extra I1PP2A. Since this is contradictory to the other results, too much I1PP2A may have been added to the sample to give informative results and may have simply drowned out the effects of the inhibitors. This experiment should also be repeated with different concentrations of added I1PP2A to determine if this is the case. FUTURE WORK From this batch of aptamers which has shown to be selective to I1PP2A, individual aptamers will be selected and sequenced. These particular sequences will be tested individually using the PP2A activity assay performed earlier to determine which single aptamer works best and under what ranges of concentrations it does so. If successful in screening a single aptamer that binds to I1PP2A with high specificity and affinity and shows promising pharmacological attributes, we will incorporate this oligonucleotide into a r AAV gene transfer vector for surgical delivery into the brain of adult rat s. PP2A activity assays from these brain tissue extracts will determine the effect these aptamers have on enzymes in vivo If the aptamer vectors are successful in increasing PP2A activity, they will be surgically delivered to the brains of disease pathology will be monitored, and spatial task acquisition and retention tests will be done on the transfected rats This will test the final hypothesis that inhibiting I1PP2A and increasing dephosphorylation by PP2A will ameli orate the symptoms of tauopathy. A CKNOWLEDGMENTS Many thanks to Dr. Aaron Hirko and Marjorie Chow for all their guidance and collaboration and to Dr. Michael King for trusting me with such a meaningful project. I would also like to thank the UF Center for Undergraduate Research and the University Scholars Program for their funding a nd support. REFERENCES  Lee VM, Trojanowski JQ. Progress from Alzheimer's tangles to pathological tau points towards more effective therapies now. J Alzheimers Dis 2006;9(3 Suppl):257 62. [3 ] Binder LI, Guillozet Bongaarts AL, Garcia Sierra F, Berry RW. Tau, tangles, and Alzheimer's disease. Biochim Biophys Acta 2005 Jan 3;1739(2 3):216 23. [4 ] Iqbal K, Alonso Adel C, Chen S, Chohan MO, El Akkad E, Gong CX, Khatoon S, Li B, Liu F, Rahman A, Tanimukai H, Grundke Iqbal I. T au pathology in Alzheimer disease and other tauopathies. Biochim Biophys Acta 2005 Jan 3;1739(2 3):198 210. [5 ] Buee L, Bussiere T, Buee Scherrer V, Delacourte A, Hof PR. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brai n Res Brain Res Rev. 2000 Aug;33(1):95 130. [6 ] Rankin CA, Sun Q, Gamblin TC. Pseudo phosphorylation of tau at Ser202 and Thr205 affects tau filament formation. Brain Res Mol Brain Res 2005 Jul 29;138(1):84 93. [7 ] Tsujio I, Zaidi T, Xu J, Kotula L, Grundke Iqbal I, Iqbal : Inhibitors of protein phosphatase 2A from human brain structures, immunocytological localization and activities towards dephosphorylation of the Alzheimer type hyperphosphorylated tau. FEBS Lett 2005 Jan 17;579(2):363 72. [8 ] Jan ssens V, Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 2001 Feb 1; 353(Pt 3): 417 39. [9 ] Lechward K, Awotunde OS, Swiatek W, Muszynska G. Protein phosphata se 2A: variety of forms and diversity of functions. Acta Biochim Act a. 2001;48(4):921 33 [10 ] Gong CX, Shaikh S, Wang JZ, Zadi T, Grundke Iqbal I, Iqbal K: Phosphatase activity toward abnormally phosphorylated tau: decrease in Alzheimer disease brain. J Neurochem 1995, 65:732 738. [11 ] Iqbal K, Alonso Adel C, El Akkad E, Gong CX, Haque N, Khatoon S, Pei JJ, Tanimukai H, Tsujio I, Wang JZ, Grundke Iqba I. Alzheimer neurofibrillary degeneration: therapeutic targets and high throughput assays. J Mol Neurosci 2003;20 (3):425 9. [12 ] Tanimukai H, Grundke Iqbal I, Iqbal K. Up regulation of inhibitors of protein phosphatase 2A in Alzheimer's disease. Am J Pathol 2005 Jun;166(6):1761 71. [13 ] Nogales E, Wang HW. Structural intermediates in microtubule assembly and disas sembly: how and why? Curr Opin Cell Biol 2006 Apr;18(2):179 84.
MATTHEW D ISAACSON D R A ARON C H IRKO AND DR M ICHAEL A K ING University of Florida | Journal of Undergraduate Research | Volume 13 Issue 3 | Summer 2012 8 [14 ] Jayasena SD: Aptamers: a n e merging c lass of m olecules t hat r ival a ntibodies in d iagnostics. Clinical Chemistry 1999;45( 9 ):1628 1650 [15 ] Mallikaratchy P, Stahelin RV, Cao Z, Cho W, Tan W. Selection of DNA ligands for protein kinase Cdelta. Chem Commun (Camb). 2006 Aug 14;(30):3229 31. [16 ] Mosing RK, Mendonsa SD, Bowser MT. Capillary e lectrophoresis SELEX s election of a ptamers with a ffinity for HIV 1 r everse t ranscriptase. Anal. Chem. 2005 Sept; 77 (19):6107 12. [17 ] Jones LA, Clancy LE, Rawlinson WD, White PA. High a ffinity a ptamers to s ubtype 3a h epatitis C v irus p olymerase d isplay g enotypic s pecificity. Antimicrobial Agents and Chemotherapy 2006 Sept;50(9)3019 27. [18 ] Murphy MB, Fuller ST, Richardson PM, Doyle SA. An improved method for the in vitro evolution of aptamers and applications in protein detection and purification Nucleic Acids Res 2003 Sept 15; 31 (18) :e110. [19 ] Owens RA. Second generation adeno associated virus type 2 based gene therapy systems with the potential for preferential integration into AAVS1. Curr Gene Ther 2002 May;2(2):145 59. [20 ] Mandel RJ, Burger C. Clinical trials in neurological disorders using AAV vectors: promises and challenges. Curr Opin Mol Ther 2004 Oct;6(5):482 90.
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