(PDF) Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery ORIGINAL ARTICLESupercoiled Minivector DNA resists shear forces associatedwith gene therapy deliveryDJ Catanese Jr1,2,3, JM Fogg1,2,3, DE Schrock II1,4, BE Gilbert1and L Zechiedrich1,2,3,4Supercoiled DNAs varying from 281 to 5302 bp were subjected to shear forces generated by aerosolization or sonication. DNAshearing strongly correlated with length. Typical sized plasmids (X3000 bp) degraded rapidly. DNAs 2000–3000 bp persistedB10 min. Even in the absence of condensing agents, supercoiled DNA o1200 bp survived nebulization, and increased forcesof sonication were necessary to shear it. Circular vectors were considerably more resistant to shearing than linear vectors of thesame length. DNA supercoiling afforded additional protection. These results show the potential of shear-resistant MinivectorDNAs to overcome one of the major challenges associated with gene therapy delivery.Gene Therapy (2012) 19, 94–100; doi:10.1038/gt.2011.77; published online 2 June 2011Keywords: Minivector DNA; Minicircle; hydrodynamic shear; nebulized; aerosolized; lung deliveryINTRODUCTIONThere are currently many challenges to gene therapy and most involvehurdles with nucleic acid delivery. Although viruses are highly effectiveand deliver high doses, they can trigger immunological and inflam-matory responses and can potentially misregulate endogenous genes.Such responses may result in disease or even death.1–4 Because of theseproblems, effort has been expended toward the development ofeffective non-viral vectors.5Plasmid DNA vectors are relatively inex-pensive, easy to make and store, and have tremendous design capacity.A shortcoming of plasmids is their size (typically 44000 basepairs (bp)), which makes them highly susceptible to shear-induceddegradation.6,7Another important challenge of gene therapy is targeting the vectorto the diseased organ. The lungs are amenable to gene therapy becausethey are accessible through the nose and mouth. Nebulization to createan aerosol is routinely used to deliver drugs for the treatment of lungdiseases. Although toxicity is lessened when plasmid DNA is deliveredby aerosol compared with systemic delivery,8,9 plasmid DNA stillcannot be delivered by this method because of extensive degrada-tion.10,11 DNA degradation during nebulization is thought to mainlyoccur because of hydrodynamic shear,12 which is the force exerted onthe DNA by the rapid flow of the solvent.DNA degradation by hydrodynamic shear was first studied over 50years ago by forcing bacteriophage DNA through a hypodermicneedle.13 Although several studies since have attempted to determinethe mechanisms behind DNA shearing, it is still not fully understoodwhat causes the DNA to degrade.7Many vehicle systems have beendeveloped to try to protect fragile plasmid vectors (or synthetic smallinterfering RNA) from delivery-associated shear forces. The cationicagents, polyethylenimine, polylysine, polyamines, and liposomes act tocondense and shield the DNA. They protect the DNA from shearingby reducing the hydrodynamic diameter of the molecule.12,14However, these vehicles are highly cytotoxic and pro-inflamma-tory,15,16 and only partially alleviate DNA shearing.17,18For the few cases in which the effect of DNA size has been studied,shear force resistance is inversely correlated with DNA length;13 000bp plasmids were more resistant to shearing than 20000 or29 000 bp plasmids measured in a rotary disk shearing device6and9800 bp plasmids sheared three times more rapidly than 5000 bpplasmids in a jet nebulizer.12 It is reasonable to extrapolate that shorterDNA vectors should be less vulnerable to shearing, but this idea couldnot be tested previously because it was not possible to generate shortercircular DNA (o2000 bp) in sufficient quantities to do the experi-ments. Because of the need for a bacterial origin of replication and aselectable Marker, thousands of bp are required for the propagationof plasmids.5Deleting such sequences, through site-specific recombi-nation, results in DNA vectors that are less toxic and less likely tobe silenced than plasmids.19,20 Still thousands of bp in length,however, these ‘Minicircles’ remain highly susceptible to shear-induced degradation.A potential way to make smaller DNA vectors is to ligate linearDNAs in vitro. Depending on sequence, shortening DNA makes itincreasingly difficult to bend into a circle because of inherentrigidity.21–23 Thus, obtaining reasonable quantities of circles lessthan 1000 bp is difficult; microgram quantities were previously thebest achievable yields.24 Manipulating DNA supercoiling throughtopoisomerase mutations and using site-specific recombination inEscherichia coli, we generate Minivectors as small as B250 bp inmilligram quantities.25,26 Nearly devoid of bacterial sequences, Mini-vectors can be used to express short hairpin RNA (shRNA), micro-RNA (miRNA) or genes in various cell types, including cells that arerefractory to plasmid transfection.26 In this paper, we use Minivectorsto determine the role of DNA length, circularity, and supercoiling onshear force resistance. We find that extrapolation from the previousReceived 10 January 2011; revised 4 March 2011; accepted 9 March 2011; published online 2 June 20111Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA; 2Verna and Marrs McLean Department of Biochemistry and MolecularBiology, Baylor College of Medicine, Houston, TX, USA; 3Department of Pharmacology, Baylor College of Medicine, Houston, TX, USA and 4University of Texas MD AndersonCancer Center School of Health Sciences, Houston, TX, USACorrespondence: Dr L Zechiedrich, Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Mail-stop: BCM-280, Houston, TX 77030,USA. E-mail: elz@bcm.eduGene Therapy (2012) 19, 94–100 2012 Macmillan Publishers Limited All rights reserved 0969-7128/12www.nature.com/gt data would not have predicted the behavior of very small, supercoiledcircles when subjected to shear forces.RESULTSShearing as a function of DNA lengthThere are multiple ways to generate hydrodynamic shear: passagethrough a narrow gauge needle, circulation through high-performanceliquid chromatography tubing, nebulization, and sonication.27 Weused nebulization because it is both highly reproducible and clinicallyrelevant. In a Collison-like jet nebulizer,28 it is far easier to collectsamples from the reservoir, and DNA degradation was identical whensampled from the aerosol or the reservoir (Supplementary Figure 1).For the experiments described below, therefore, we sampled from thereservoir.Supercoiled DNAs varying from 281 to 5302 bp (Table 1) weresubjected to nebulization and their survival was analyzed by gelelectrophoresis (Figure 1). The extent of DNA shearing was quantifiedby measuring the disappearance of intact DNA vector over time. Theability to withstand shear forces in the nebulizer was, in general,inversely correlated with DNA length. Supercoiled DNA vectors42600bp, the size range of DNA vectors used previously for delivery,fragmented completely and rapidly and displayed pseudo-exponentialdecay (Figure 1b). The sigmoid shape of the decay curves for super-coiled DNA vectors between 1580 and 2232bp showed an initialresistance to shearing followed by degradation (Figure 1b), a verydifferent behavior from that of the larger vectors. In common, allDNA vectors sheared into fragments that ranged from 200 to 1000bp(Figure 1a). Supercoiled Minivectors p1243 bp slightly concentratedwith time in the nebulizer because of evaporation (Figure 1b). Toaddress whether changing DNA concentrations could affect shear forcesurvival, solutions of 1873 bp plasmid ranging from 1 to 10 mgml1were nebulized. The decay kinetics were identical at all concentrations(data not shown).To facilitate comparisons among the differently sized DNA vectors,we introduce a term, Survival50, which is the time at which 50% ofintact vectors remain. Nebulization Survival50 as a function of DNAvector length is shown (Figure 1c). Comparing the data in this wayrevealed important and unexpected results. First, Survival50 did notdepend monotonically on DNA length, but instead exhibited a thresh-old-like behavior as defined by the asymptote. Below this threshold,which may reflect a critical hydrodynamic diameter, DNA vectors didnot appreciably shear. Second, Minivectors survived better thanplasmids of the same length.There are several differences between plasmids and Minivectors.Plasmids contain origins of replication and genes encoding antibioticresistance; Minivectors contain attR.25 In addition, different bacterialstrains are used for the propagation of plasmids and Minivectors.25,26As a consequence, Minivectors have increased supercoiling(sB0.09 compared with sB0.075 for plasmids29), which shouldfurther decrease their hydrodynamic diameter, and this may explaintheir increased protection from shear forces. To address the potentialeffect of increased supercoiling, we propagated a plasmid or aMinivector of the same length in LZ54,30 the bacterial strain used togenerate Minivectors. The 1711bp plasmid, pDJC1, propagated inLZ54 persisted as long as the 1714bp Minivector (Figure 1c, compareopen circle with closed circle, arrow). Plasmid pQR499 (1873 bp)isolated from LZ54 also persisted longer than when it had beenpropagated in DH5a(Figure 1c, arrow). Increased supercoiling,therefore, and not differences in sequence between plasmids andMinivectors, accounted for the increased shear resistance.The advantages of reducing vector size were even more pronouncedwhen Survival90, the time at which 90% of vectors remained, wasTable 1 DNA vectors used in this studyLength (bp) DNA vector EncodesaNebulization Survival50 (min) Sonication Survival50 (min) Reference or source281 mv281 — 430 ND 25336 mv336 — ND 96.13±2.95 This study383 mv-H1-GFPshRNA shRNA to eGFP 430 ND 26562 Dimer of mv281 — 430 ND 25672 Dimer of mv336 — 430 21.86±3.68 This study985 mv-H1-miR31 Human miR-31 430 ND This study999 mv999 — ND 5.98±1.46 251109 mv-KB4TAL-GLuc Gaussia luciferase 430 ND This study, Nanolight Technology1243 mv-KB4TAL-mCherry mCherry 430 3.73±0.55 This study531580 mv-CMV-GLuc Gaussia luciferase 32.3±3.1 1.77±0.69 This study, Nanolight Technology1711 pDJC1 — 21.6±3.228.2±3.4bND This study1714 mv-CMV-mCherry mCherry 27.5±2.3 ND T his study531873 pQR499 — 22.0±1.124.4±1.3b0.85±0.10 542067 pINV2067 — 19.1±0.2 ND Invitrogen supercoiled DNA ladder2232 pAO — 16.6±3.5 ND 552679 mv-CMV-Luc2 Firefly luciferase 9.6±1.5 ND T his study, Promega2686 pUC18 — 10.8±1.4 ND Novagen Inc.3000 pBLUESCRIPT — 6.1±0.1 ND Stratagen e3459 pJB3.5i attB,attP 3.4±0.9 ND 563869 pMC339-BbvCI attB,attP 4.0±0.5 0.17±0.04 255302 pCR2.1-norE norE 1.5±0.1 ND This studyAbbreviations: eGFP, enhanced green fluorescent protein; miR, microRNA; ND, not determined; shRNA, short hairpin RNA.aAll Minivectors contain attR;25 all plasmids encode bla.bPropagated in LZ54.DNA shearingDJ Catanese Jr et al95Gene Therapy considered (Figure 1c, inset). Below 3000bp, there was a very steepinverse correlation between DNA length and Survival90 values. ForDNA vectors o2000 bp, Survival90 values were longer than 10min, asa result of the initial resistance to shearing and as seen by thesigmoidal decay. Considering various percentages of DNA vectorsurvival might be useful for calculating the relative benefits andrisks of using a particular DNAvector length (Supplementary Table 1).Effect of DNA circularity and supercoiling on shear force resistanceTo better understand the contribution of DNA supercoiling to shearforce resistance, we compared linear, nicked and relaxed forms of thevectors (Figure 2). Half of the negatively supercoiled 1873 bp plasmid,pQR499, survived 22min, longer than either the relaxed, open-circular (17.5 min) or nicked (17.2 min) forms of the vector. Thenearly identical Survival50 values of the nicked and relaxed plasmidsrevealed, somewhat unexpectedly, that a single-strand break does notsignificantly weaken this vector during nebulization. Circularityhad a bigger impact on Survival50 than supercoiling did; half of thelinear form of the 1873 bp DNA was sheared in B4 min. The fragilityof linear DNA during nebulization was the same whether the endswere blunt, had 2-base 3¢-overhangs or had 4-base 5¢-overhangs (datanot shown); therefore, the nature of the ends did not affect DNAshearing.Because shorter vectors completely survived nebulization, we usedsonication to determine how supercoiling affected their shearing. Likenebulization, sonication also generates highly reproducible shearforces, but these forces can be much greater and can be generatedfor longer periods of time. The difference in the shear force magnitudeis exemplified by the differences in Survival50 values between thesupercoiled 3869 bp plasmid (sonication Survival50¼0.17min andnebulization Survival50¼4 min) and the 1873 bp plasmid (sonicationSurvival50¼0.85 min and nebulization Survival50¼22 min). Despitethe difference in shear force magnitude, the two methods for shearingyielded the same general result—a strong correlation of DNA vectorlength and shearing (Figure 3). For example, the 3869 bp plasmidwas sheared 4500 times more rapidly (Survival50¼0.17 min) than avector approximately one-tenth that length, the 336 bp Minivector(Survival50¼96 min).Figure 1 DNA survival during nebulization as a function of DNA length. (a) Representative gels of DNA vectors subjected to nebulization as sampled fromthe reservoir. (b) Graphical representation of DNA shearing as a function of length (averaged for at least three separate experiments). The curves representthe fit to a sigmoidal function (Supplementary Table 1). Minivectors p1243 bp were not fitted because after 30min the intact DNA did not decrease.Nebulization survival times were determined for each DNA vector. (c) The time at which 50% of the vector survived (Survival50). Each Survival50 value is themean from at least three separate experiments. Error bars represent the standard deviation. The arrows denote Survival50 of the two plasmids, pDJC1 andpQR499, that were propagated in LZ54. Minivectors are represented by open circles and plasmids are shown as closed circles. The inset shows Survival90values, which is the time at which 90% of vectors remained.DNA shearingDJ Catanese Jr et al96Gene Therapy It was impossible to distinguish Survival50 by sonication for thecircular forms of the 1873 bp plasmid, which were all about a minute,but the value for the linear form was only 0.2 min. Although the DNAvectors could be subjected to sonication much longer than they couldto nebulization, sonicating DNA for more than 90min is impractical.Therefore, to determine how DNA supercoiling affected shear forcesurvival required a balance between a Minivector large enough fordifferences to be detected and small enough for those differences to beseparated. A 999bp Minivector fit these criteria. Like the 1873bpplasmid sheared by nebulization, this Minivector sheared according toits topology; linear sheared with a sonication Survival50¼0.8 min,nicked¼3.8 min, relaxed¼4.6 min and supercoiled¼6min.With regard to shear force resistance, circularity is the mostimportant property of a DNA vector for protection. This protectionis B4- to 6-fold when comparing the nicked/relaxed and linear formsof each vector (Figure 2). Supercoiling provides additional protectionfrom shear forces. The supercoiled 999 and 1873bp DNA vectorspersisted 58 and 28% longer, respectively, than their nicked counter-parts (Figure 2).DISCUSSIONModel for DNA shearingEarly studies of DNA shearing showed that the hydrodynamic shearforce exerted on a DNA molecule increases with the square ofthe hydrodynamic diameter (the length-dependent diameter of theDNA molecule in the fluid stream).12,31 Our data are consistent withthis relationship. Circularity, decreasing length and increasing super-coiling, properties that we find to protect DNA vectors from shearing,should all reduce hydrodynamic diameter. Intuitively, the hydrody-namic diameter of a circular DNA vector should be approximately halfthe length of the linear form,7and this idea is supported by experi-mental data.32 Our findings that the 1873bp linear DNA sheared asrapidly as the 3869 bp supercoiled plasmid and that the linear 999 bpDNA sheared comparably to a plasmid roughly twice its length isconsistent with this concept. In addition, our observation that super-coiling affords additional protection is supported by the increasedsurvival of supercoiled DNA vectors compared with the nicked orrelaxed forms of the same length.Because smaller DNA vectors experience less force, they survivelonger, but what finally makes them break? If single-stranded breakswere generated, we would expect to see a transient appearance ofnicked intermediate as supercoiled DNA is sheared. Nicked DNA,however, was not observed during shearing of the supercoiled vectors,indicating that shearing generates primarily double-stranded breaks.The absence of any full-length linear degradation products indicatesthat following the initial double-strand break, additional breaks occurin rapid succession, consistent with the extremely rapid conversion oflinear vectors to smaller fragments.The pronounced lag observed before the smaller DNA vectorsdegraded indicates that the shear forces in the nebulizer must notinitially be harsh enough to break these smaller vectors, and that thecontinuous re-circulation of the DNA vectors may stretch or contortthem into a conformation that is then more susceptible to shearing.There are two proposed competing effects of DNA supercoiling onshear survival: torsional strain, which might make the molecule moresusceptible to shearing, and DNA compaction, which should make themolecule more resistant to shearing.7Although our data clearly showthat the beneficial effect of compaction is the dominant effect,torsional stress-induced deformation of DNA33–35 may contribute tothe eventual shearing of the vectors, thereby counteracting theprotective effect of supercoiling-mediated DNA compaction. As theFigure 2 Effect of DNA topology on DNA survival. (a) How topology of plasmid DNA (1873 bp) influences its survival during nebulization. The fraction ofDNA vector of each topology over time (averaged from at least three experiments) is shown. The curves are shown fitted to a sigmodial function. (b)Howtopology of Minivectors (999bp) influences its survival during sonication. The fraction of DNA was quantified the same as in (a).Figure 3 DNA survival during sonication as a function of DNA length.Because more force was necessary to degrade the smaller Minivectors,sonication was employed. Sonication survival times were determined foreach DNA vector in the same manner as for nebulization in Figure 1. Theplot shows the time at which 50% of each vector survived (Survival50)versus its length. Each Survival50 value is the mean from at least threeseparate experiments. Error bars represent the standard deviation.DNA shearingDJ Catanese Jr et al97Gene Therapy reservoir volume decreases, the rate of recirculation increases(Supplementary Figure 2), consequently the frequency of exposureto shear forces increases, and there may be less time for DNA torecover from shear force-induced contortion. The stretched, contortedand weakened DNA is then sheared more readily. This model predictsthat DNA base pair steps that are more likely to contort would be theweak points of the molecule that shear. It is possible that thenebulization protocol during treatment of a patient could be alteredto decrease this frequency of recycling, perhaps as simply as bymaintaining a constant reservoir volume.Therapeutic consequences of DNA shearingThe most obvious detriment of DNA shearing is a reduction in theamount of intact vector remaining for therapy. Larger DNA vectorscompletely degrade into small fragments over the course of nebuliza-tion. These fragments are presumably unable to elicit a therapeuticeffect as the transgene or shRNA together with the promoter aredestroyed. It might be tempting to increase the DNA dose tocompensate for this loss. Doing so, however, could have negativeconsequences. First, increasing the amount of DNA vector would alsorequire a commensurate increase in the amount of toxic deliveryvehicle. Second, delivery of increased degraded DNA fragments couldinduce DNA repair and recombination pathways or trigger apoptosis.Third, cells could randomly ligate the fragmented DNA. Althoughthere is the potential of the ligations forming large episomal con-catemers that could persist to cause stable long-term transgeneexpression,36 there is also the possibility that the randomly ligatedDNA fragments could join together to generate new, potentially toxicsequences. One way to avoid these problems would be to haltnebulization earlier. For example, DNA vectors o2000 bp, for whichSurvival90 values were greater than 10 min, could be halted after10 min. Shortening nebulization treatments would not be necessaryfor the smaller Minivectors (o1000 bp) because very little shearingoccurs, even after 30 min.DNA vector delivery to the lungsIdentifying DNA vector lengths that survive shear forces has importantimplications for gene therapy delivery no matter what the deliveryroute; however, the use of a Collison-like jet nebulizer makes our dataparticularly germane for the consideration of DNA delivery to thelungs. The lungs are readily accessible by intratracheal, intranasal andaerosol delivery methods, and any of these routes is amenable to DNAvector delivery. Aerosol delivery, in particular, is non-invasive, deliversdirectly to the affected tissue and may help prevent off-target com-plications. In addition, aerosol delivery allows DNA to be delivered tothe lungs in much higher quantities than systemic administration.37A number of promising gene therapy targets have been identifiedfor the treatment of pulmonary diseases. Dozens of clinical trials areongoing that target, for example, cystic fibrosis (http://www.wiley.com/legacy/wileychi/genmed/clinical/). Disappointingly, however,these existing therapies have so far been unsuccessful.38 Asthma as adisease target has high potential for therapeutic RNA interference,including shRNA39 and miRNA,40 but the bottleneck for success fortreatment of asthma is the same as for other diseases—delivery of thenucleic acid therapy. The observation that Minivectors can overcomesome of the most difficult obstacles associated with delivery renewsenthusiasm for gene therapy for the lungs.Additional therapeutic benefits of small, supercoiled DNA vectorsIn a previous study from our group, Minivectors of 383 bp, encodingan shRNA against a pathogenic protein in lymphoma, survived intactin human serum for 448h, whereas synthetic small interfering RNAand plasmids were degraded within a couple of hours.26 Certain DNAsequences, such as polyA signals and antibiotic resistance genes thatare commonly found in DNA vectors, are particularly susceptible toattack by nucleases.41 Some of the biostability of Minivectors in theserum may be a consequence of such sequences being absent from theMinivectors.Extra DNA on traditional plasmids contain bacterial sequences thatinduce immunotoxic responses, primarily because of immuneresponses to CpG motifs42 that are approximately four times moreprevalent in bacterial than mammalian DNA.43 In addition to toxicity,bacterial sequences also induce transcriptional silencing of episomaltransgenes.44 ‘Minicircles’ that lack bacterial sequences exhibit sig-nificantly increased transgene expression.20,44,45In addition to increased shear force survival, reducing the length ofDNA vectors also improves cell transfection.26,46,47 Minivectors of afew hundred bp transfect cells that are refractory to transfection withconventional plasmids.26 The smaller size of Minivectors means morecopies per unit mass. This fact, coupled with the very high transfectionefficiency,26 suggests that a lower DNA vector dose and, consequently,less toxic transfection vehicle may be used.Our data show that survival of shear forces associated withnebulization requires DNA vectors to be circular and shorter than2000 bp. Although it may seem that therapeutically useful Minivectorsmay express only shorter sequences, such as shRNA, miRNA and smallgenes, in fact we have not lost the ability to express larger genes. Largergenes can be split into multiple smaller segments that when expressedwill reconstitute in vivo to form a functional protein. The concept ofsplitting genes has been studied for many decades, and one of the firstreports involved splitting the gene encoding b-galactosidase.48 Otherexamples of genes that have been split and reconstituted in vivoinclude the genes encoding ubiquitin,49 PurN,50 adenylate kinase51and the yellow fluorescent protein, Venus.52 Because they are nearlydevoid of bacterial sequences, almost all of the Minivector is availableto encode a useful sequence. Even at 2000 bp, Minivectors can encodeapromoterandB1500 bp of gene sequence (either an intact smallgene or gene fragment), significantly increasing the arsenal of usefultherapeutic sequences.To our knowledge, no previous study has investigated such a broadrange of DNA sizes and topologies as reported here. These data will beuseful for identifying the factors most important for resisting shearforces involved in gene therapy delivery. Knowing how to mitigateDNA vector degradation will facilitate the development of new, moreefficacious gene therapy vectors. In addition, the use of the nebulizermakes our data particularly germane to clinical applications. Theability of Minivectors to resist shear forces associated with genetherapy delivery renews hope of treating human diseases using DNAvectors.MATERIALS AND METHODSChemicals, reagents and equipmentAcrylamide was from EMD Chemicals (Gibbstown, NJ, USA), agarose fromISC BioExpress (Kaysville, UT, USA) and SYBR Gold from Invitrogen(Hercules, CA, USA). Restriction enzymes were from New England Biolabs(Ipswich, MA, USA). All other chemicals were from Fisher Scientific (Waltham,MA, USA). Plasmid Maxi kit was from Qiagen (Valencia, CA, USA) andAmicon Ultra centrifugal filters were from Millipore (Billerica, MA, USA). TheAerotech II jet nebulizer was from CSI-US Inc. (Bedford, MA, USA) and theAridyne 2000 compressor was from Allied Healthcare Products (St Louis, MO,USA). The 1/8in probe sonicator (Model 60 Sonic Dismembrator) was fromFisher Scientific. PC Image and Total Lab software were from FotodyneDNA shearingDJ Catanese Jr et al98Gene Therapy (Hartland, WI, USA) and Total Lab (Durham, NC, USA), respectively.KaleidaGraph (version 4.1) was from Synergy Software (Reading, PA, USA).DNA generation and manipulationDNA vectors used in this study are listed in Table 1. Throughout the text, werefer to these by their length. Following the convention of using ‘p’ in front ofplasmid names, we designate Minivectors with ‘mv’. Parent plasmids used togenerate Minivectors are designated ‘pMV’. pMV-KB4TAL-GLuc and pMV-CMV-GLuc were gifts from Dr Stephen Gottschalk (Baylor College of Medi-cine, Houston, TX, USA) and pMV-CMV-Luc2 was a gift from Dr DavidSpencer (Baylor College of Medicine). pMV-H1-miR31, pMV-KB4TAL-mCherry and pMV-CMV-mCherry were gifts from Dr Martin M Matzukand Dr Zhifeng Yu (Baylor College of Medicine). pQR499 was a gift fromDr John Ward (University College London, London, UK). pDJC1 was con-structed by digesting pQR499 with TfiIandAflIII. The recessed ends of thedigested pQR499 were filled in with T4 DNA polymerase and subsequentlyligated with T4 DNA ligase. Plasmids were generated in Escherichia coli DH5acells, isolated using a Plasmid Maxi kit as per the manufacturer’s instructions, andsubsequently desalted and concentrated using Amicon Ultra centrifugal filters.Minivectors were obtained as follows. Minivector parent plasmids weretransformed into E. coli strain LZ54.30 Large-scale lInt-mediated recombina-tion and Minivector isolation was performed as described.25,26 To generatenicked DNA vector, nicking endonuclease Nt.BbvCI was used following themanufacturer’s protocols. To obtain relaxed DNA, nicked DNA was religatedusing T4 DNA ligase. Linearization was performed with either BspHI, EcoRV,PvuIorScaI, depending on which overhang end was desired, as per themanufacturer’s protocols. Nicked, relaxed and linear DNAs were extracted withphenol:chloroform:isoamyl alcohol (25:24:1), extracted with chloroform, andprecipitated with ethanol. Each DNA topology was resuspended in TE buffer(10 mMTri s -HCl , 1 mMEDTA, pH 8) and verified using gel electrophoresisbefore nebulization or sonication.DNA shearingFor nebulization, 10 ml of DNA at 1 mgml1in TE buffer was added to anAerotech II jet nebulizer. Air was delivered to the nebulizer at a rate of10 l min1and gauge pressure of 50p.s.i. by an Aridyne 2000 compressor.For the studies of the effects of DNA length on nebulization survival, 15 mlaliquots were removed from the nebulizer reservoir before and at intervalsthroughout nebulization up to 30 min, at which point the DNA solution wasdepleted. Because of the dramatic changes that occurred early, aliquots weretaken at 1 min intervals initially and at 2 min intervals after 10 min.For sonication, 1 ml of DNA at 1 mgml1in TE buffer in a 1.7ml eppendorftube was incubated on ice during sonication with a 1/8 in probe sonicator atsetting 5, which has an output of 3–7W (root mean square). For consistency,the probe was inserted halfway down into the solution and the sides of the tubewere avoided. 15 ml aliquots were removed before sonication and at the timepoints indicated.All DNA shearing experiments were performed a minimum of three separatetimes. DNA was analyzed by electrophoresis on 1% agarose gels for 41000 bpor 2% agarose gels or 5% acrylamide (29:1 acrylamide:bis-acrylamide) gels foro1000 bp in 40 mMTris-acetate and 2 mMEDTA. All gels were submitted to125 V for 2 h, stained with SYBR Gold for 20 min and visualized using PCImage. Total Lab was utilized to quantify the remaining intact DNA. Data wereanalyzed and fitted using KaleidaGraph.CONFLICT OF INTERESTDr Jonathan Fogg and Dr Lynn Zechiedrich are co-inventors on an issuedpatent, and together with Dr Daniel Catanese and Dr Brian Gilbert, are also co-inventors on another patent application. These patents cover the Minivectortechnology used in the work reported in this paper.ACKNOWLEDGEMENTSWe thank Alexander Seryshev and Alex J Brewer III for technical assistance,Dr David B Corry and Dr Peter Hu for helpful discussions, and Dr John Ward,Dr Stephen Gottschalk, Dr David Spencer, Dr Martin M Matzuk and DrZhifeng Yu for reagents. DJC was funded by Infection and Immunity TrainingGrant T32 AI55413-05. DES was supported by the Molecular GeneticTechnology Program at the University of Texas MD Anderson Cancer Center.BEG was supported by the Clayton Foundation for Research. This workwas supported by a grant from the National Institutes of Health RO1A1054830to LZ. This paper is dedicated to Margie Catanese, Frank Fogg, RosemaryCmarik, Sissy (Zechiedrich) James, and all who have lost the battle againstlung cancer.1 Somia N, Verma IM. Gene therapy: trials and tribulations. Nat Rev Genet 2000; 1:91–99.2 Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch Pet al. LMO2-associated clonal Tcell proliferation in two patients after gene therapy forSCID-X1. Science 2003; 302: 415–419.3 Couzin J, Kaiser J. Gene therapy. As Gelsinger case ends, gene therapy suffers anotherblow. Science 2005; 307:1028.4 Kaiser J. Gene t herapy. Panel urges limit s on X-SCID trial s. 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Split ubiquitin as a sensor of protein interactions in vivo.Proc Natl Acad Sci USA 1994; 91: 10340–10344.50 Ostermeier M, Nixon AE, Shim JH, Benkovic SJ. Combinatorial protein engineering byincremental truncation. Proc Natl Acad Sci USA 1999; 96: 3562–3567.51 Nguyen PQ, Liu S, Thompson JC, Silberg JJ. Thermostability promotes the co-operative function of split adenylate kinases. Protein Eng Des Sel 2008; 21:303–310.52 Hoff KG, Culler SJ, Nguyen PQ, McGuire RM, Silberg JJ,Smolke CD. In vivo fluorescentdetection of Fe–S clusters coordinated by human GRX2. Chem Biol 2009; 16:1299–1308.53 Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. NatMethods 2005; 2:905–909.54 Stuchinskaya T, Mitchenall LA, Schoeffler AJ, Corbett KD, Berger JM, Bates AD et al.How do type II topoisome rases use ATP hydrolysis to simplify D NA topology beyondequilibrium? Investigating the relaxation reaction of nonsupercoiling type II topoisome-rases. J Mol Biol 2009; 385: 1397–1408.55 Wu HY, Liu LF. DNA looping alters local DNA conformation during transcription. JMolBiol 1991; 219: 615–622.56 Bliska JB, Cozzarelli NR. Use of site-specific recombination as a probe of DNAstructure and metabolism in vivo.JMolBiol1987; 194: 205–218.This work is licensed under the Creative CommonsAttribution-NonCommercial-No Derivative Works 3.0Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)DNA shearingDJ Catanese Jr et al100Gene TherapySupplementary resource (1)Supplementary InformationDataJune 2011Daniel J Catanese · J M Fogg · D E Schrock · B E Gilbert · L ZechiedrichCitations (50)References (66)... There are several types of non-viral minimized DNA vectors in pre-clinical use (reviewed in [1]). Here, we will highlight recent advances for minicircles [1,7,8] and minivectors [1,9,10]. Several different methods exist for the production of these vectors [7,8,11], but common to most is the use of bacteria to propagate plasmids. ...... One key difference between the two is that minivectors employ a more rigorous purification method that takes advantage of the small size of the minivectors generated, allowing for complete removal of the larger miniplasmid contaminant. Additional advantages include increased negative supercoiling and the ability to generate vectors as small as a few hundred base pairs [9,10]. ...... With diameters of around 40 nm [83], supercoiled minivectors can be made small enough such that their diameter is within a nanoparticle size range [10,83]. Furthermore, when complexed with delivery vehicle, for example, poly-L-lysine-polyethylene glycol, minivectors are highly homogenous, monodisperse, and adopt a needle-shaped conformation; comparatively, plasmids are not nanoparticle-sized and adopt highly heterogeneous shapes (Figure 1). ...Improving therapeutic potential of non-viral minimized DNA vectorsArticleFull-text availableOct 2020 Lirio Milenka Arevalo-Soliz Cinnamon HardeeJonathan M FoggLynn ZechiedrichThe tragic deaths of three patients in a recent AAV-based X-linked myotubular myopathy clinical trial highlight once again the pressing need for safe and reliable gene delivery vectors. Non-viral minimized DNA vectors offer one possible way to meet this need. Recent pre-clinical results with minimized DNA vectors have yielded promising outcomes in cancer therapy, stem cell therapy, stem cell reprograming, and other uses. Broad clinical use of these vectors, however, remains to be realized. Further advances in vector design and production are ongoing. An intriguing and promising potential development results from manipulation of the specific shape of non-viral minimized DNA vectors. By improving cellular uptake and biodistribution specificity, this approach could impact gene therapy, DNA nanotechnology, and personalized medicine.ViewShow abstract... Minicircles are a type of newly developed DNA carriers for gene therapy 22 . The main features of minicircles include the cleaner gene background with minimal viral or bacterial gene elements, sustained high-level protein expression, and more importantly, the small size that may allow the use of aerosols for drug delivery 23 . The latter may be a distinct advantage against coronaviruscaused respiratory diseases. ...High-Potency Polypeptide-based Interference for Coronavirus Spike GlycoproteinsPreprintApr 2021Jianpeng MaAdam Campos AcevedoQinghua WangThe world is experiencing an unprecedented coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 spike protein-based vaccines are currently the main preventive agent to fight against the virus. However, several variants with extensive mutations in SARS-CoV-2 spike proteins have emerged. Some of these variants exhibited increased replication, higher transmission and virulence, and were partially resistant to antibody neutralization from natural infection or vaccination. With over 130 million confirmed cases and widespread vaccination around the globe, the emergence of new escape SARS-CoV-2 variants could be accelerated. New therapeutics insensitive to mutations are thus urgently needed. Here we have developed an inhibitor based on SARS-CoV-2 spike protein that potently reduced pseudovirus infectivity by limiting the level of SARS-CoV-2 spike proteins on virion envelope. Most importantly, the inhibitor was equally effective against other coronavirus spike proteins that shared as low as 35% amino-acid sequence identity, underscoring its extreme tolerance to mutations. The small-sized inhibitor would also allow simple delivery by, for instance, nasal spray. We expect the inhibitor reported here to be an invaluable aid to help end COVID-19 pandemic. Furthermore, the use of a partial native sequence or its homologues to interfere with the functions of the native protein represents a novel concept for targeting other viral proteins in combating against important viral pathogens.ViewShow abstract... Plasmid DNAs (pDNA) which are commonly used for gene therapy may degrade upon atomization. The hydrodynamic stress generated during atomization (during spray-drying or spray-freeze drying) can shear pDNA molecules into open circular and fragmented structures [141] and the extent of shearing is dependent on the length of DNA [140]. In addition, exposure to the air-liquid interface may result in the oxidation of sugar moieties in the DNA leading to opening of DNA strand [140]. ...Dry Powder Pharmaceutical Biologics for Inhalation TherapyArticleMar 2021ADV DRUG DELIVER REV Rachel Yoon Kyung ChangMichael Y.T. Chow Dipesh Khanal Hak-Kim ChanTherapeutic biologics such as genes, peptides, proteins, virus and cells provide clinical benefits and are becoming increasingly important tools in respiratory medicine. Pulmonary delivery of therapeutic biologics enables the potential for safe and effective treatment option for respiratory diseases due to high bioavailability while minimizing absorption into the systemic circulation, reducing off-target toxicity to other organs. Development of inhalable powder formulation requires stabilization of complex biological materials, and each type of biologics may present unique challenges and require different formulation strategy combined with manufacture process to ensure biological and physical stabilities during production and over shelf-life. This review examines key formulation strategies for stabilizing proteins, nucleic acids, virus (bacteriophages) and bacterial cells in inhalable powders. It also covers characterization methods used to assess physicochemical properties and aerosol performance of the powders, biological activity and structural integrity of the biologics, and chemical analysis at the nanoscale. Furthermore, the review includes manufacture technologies which are based on lyophilization and spray-drying as they have been applied to manufacture Food and Drug Administration (FDA)-approved protein powders. In perspective, formulation and manufacture of inhalable powders for biologic are highly challenging but attainable. The key requirements are the stability of both the biologics and the powder, along with the powder dispersibility. The formulation to be developed depends on the manufacture process as it will subject the biologics to different stresses (temperature, mechanical and chemical) which could lead to degradation by different pathways. Stabilizing excipients coupled with the suitable choice of process can alleviate the stability issues of inhaled powders of biologics.ViewShow abstract... Many human clinical trials have utilized plasmid vectors to deliver transgenes [23,53]. Beyond improved transfection rates compared to plasmids, MCs are more resistant to gene silencing due to fewer CpG motifs [54], DNA shearing forces [55], possessing a higher supercoiled fraction [56], and having enhanced serum stability [57]. To facilitate use in humans, a critical aspect to optimize is the MC to transfection agent ratio, and MCs allow for a higher effective dose than plasmids because a given transfection agent amount will contain more moles of MCs than plasmids. ...A survivin-driven tumour-activatable minicircle system for prostate cancer theranosticsPreprintFull-text availableJun 2020TianDuo WangYuanxin ChenDavid Goodale John RonaldGene vectors driven by tumour-specific promoters to express reporter genes and therapeutic genes are an emerging approach for improved cancer diagnosis and treatment. Minicircles (MCs) are shortened plasmids stripped of prokaryotic sequences and have potency and safety characteristics that are beneficial for clinical translation. We previously developed survivin-driven, tumour-activatable MCs for cancer detection via a secreted blood reporter assay. Here we present a novel theranostic system for prostate cancer featuring a pair of survivin-driven MCs, combining selective detection of aggressive tumours via a urinary reporter test and subsequent tumour treatment with gene-directed enzyme prodrug therapy.MethodsWe engineered both diagnostic and therapeutic survivin-driven MCs expressing Gaussia luciferase, a secreted reporter that is detectable in the urine, and cytosine deaminase:uracil phosphoribosyltransferase fusion, respectively. Diagnostic MCs were evaluated in mice carrying orthotopic prostate tumours with varying survivin levels, measuring reporter activity in serial urine samples. Therapeutic MCs were evaluated in mice receiving prodrug using bioluminescence imaging to assess cancer cell viability over time.ResultsDiagnostic MCs revealed mice with aggressive prostate tumours exhibited significantly higher urine reporter activity than mice with non-aggressive tumours and tumour-free mice. Combined with 5-fluorocytosine prodrug treatment, therapeutic MCs resulted in reduced bioluminescence signal in mice with aggressive prostate tumours compared to control mice.ConclusionSequential use of these MCs may be used to first identify patients carrying aggressive prostate cancer by a urinary reporter test, followed by stringent treatment in stratified individuals identified to have high-risk lesions. This work serves to highlight tumour-activatable MCs as a viable platform for development of gene-based tumour-activatable theranostics.ViewShow abstractLarge BACs transfect more efficiently in circular topologyArticleAug 2021Anal BiochemYin Cheng Wong Andrew OsahorFarooq Omar Maan Al-AjliKumaran NarayananThe effect of DNA topology on transfection efficiency of mammalian cells has been widely tested on plasmids smaller than 10 kb, but little is known for larger DNA vectors carrying intact genomic DNA containing introns, exons, and regulatory regions. Here, we demonstrate that circular BACs transfect more efficiently than covalently closed linear BACs. We found up to 3.1- and 8.9- fold higher eGFP expression from circular 11 kb and 100 kb BACs, respectively, compared to linear BACs. These findings provide insights for improved vector development for gene delivery and expression studies of large intact transgenes in mammalian cells.ViewShow abstractDegradation study on molecules released from laser-based jet injectorArticleApr 2021INT J PHARMACEUTJan Krizek Barbora LavickovaChristophe MoserDevelopment of needle-free methods to administer injectable therapeutics has been researched for a few decades. We focused our attention on a laser-based jet injection technique where the liquid-jet actuation mechanism is based on optical cavitation. This study investigates the potential damage to therapeutic molecules which are exposed to nanosecond laser pulses in the configuration of a compact laser-based jet injection device. Implementation of a pulsed laser source at 1574 nm wavelength allowed us to generate jets from pure water solutions and circumvent the need to reformulate therapeutics with absorbing dyes. We performed H1-NMR analysis on exposed samples of Lidocaine and δ-Aminolevulinic acid. We made several tests with linear and plasmid DNA to assess the structural integrity and functional potency after ejection with our device. The tests showed no significant degradation or detectable side products, which is promising for further development and clinical applications.ViewShow abstractA survivin-driven, tumor-activatable minicircle system for prostate cancer theranosticsArticleFull-text availableJan 2021TianDuo WangYuanxin ChenDavid Goodale John RonaldGene vectors regulated by tumour-specific promoters to express transgenes specifically in cancer cells are an emerging approach for both cancer diagnosis and treatment. Minicircles are shortened plasmids stripped of prokaryotic sequences which have potency and safety characteristics beneficial for clinical translation. Previously, we developed minicircles driven by the tumour-specific survivin promoter, which exhibits elevated transcriptional activity in aggressive cancers, to express a secreted reporter for blood-based cancer detection. Here we present the first activatable, cancer theranostic minicircle system featuring a pair of diagnostic and therapeutic minicircles expressing either Gaussia luciferase for urine-based cancer detection, or cytosine deaminase:uracil phosphoribosyltransferase for gene directed enzyme prodrug therapy. Diagnostic minicircles revealed urinary reporter output related to cellular survivin levels. Notably, mice with aggressive prostate tumours exhibited significantly higher urine reporter activity than mice with non-aggressive tumours and healthy mice after intratumoral minicircle administration. Therapeutic minicircles displayed specific cytotoxicity in survivin-rich cancer cells and significantly attenuated the growth of aggressive orthotopic prostate tumours in mice. Use of these minicircles together creates a theranostic system that can first identify patients carrying aggressive prostate cancer via a urinary test, followed by stringent control of tumour progression in stratified individuals who carry high-risk prostate lesions.ViewShow abstractBrain Delivery of Nanomedicines: Trojan Horse Liposomes for Plasmid DNA Gene Therapy of the BrainArticleFull-text availableNov 2020 William M. PardridgeNon-viral gene therapy of the brain is enabled by the development of plasmid DNA brain delivery technology, which requires the engineering and manufacturing of nanomedicines that cross the blood-brain barrier (BBB). The development of such nanomedicines is a multi-faceted problem that requires progress at multiple levels. First, the type of nanocontainer, e.g., nanoparticle or liposome, which encapsulates the plasmid DNA, must be developed. Second, the type of molecular Trojan horse, e.g., peptide or receptor-specific monoclonal antibody (MAb), must be selected for incorporation on the surface of the nanomedicine, as this Trojan horse engages specific receptors expressed on the BBB, and the brain cell membrane, to trigger transport of the nanomedicine from blood into brain cells beyond the BBB. Third, the plasmid DNA must be engineered without bacterial elements, such as antibiotic resistance genes, to enable administration to humans; the plasmid DNA must also be engineered with tissue-specific gene promoters upstream of the therapeutic gene, to insure gene expression in the target organ with minimal off-target expression. Fourth, upstream manufacturing of the nanomedicine must be developed and scalable so as to meet market demand for the target disease, e.g., annual long-term treatment of 1,000 patients with an orphan disease, short term treatment of 10,000 patients with malignant glioma, or 100,000 patients with new onset Parkinson s disease. Fifth, downstream manufacturing problems, such as nanomedicine lyophilization, must be solved to ensure the nanomedicine has a commercially viable shelf-life for treatment of CNS disease in humans.ViewShow abstractInorganic Nanomaterial‐Mediated Gene Therapy in Combination with Other Antitumor Treatment ModalitiesArticleOct 2020ADV FUNCT MATERGuanyou Lin Richard A. ReviaMiqin ZhangCancer is a genetic disease originating from the accumulation of gene mutations in a cellular subpopulation. Although many therapeutic approaches have been developed to treat cancer, recent studies have revealed an irrefutable challenge that tumors evolve defenses against some therapies. Gene therapy may prove to be the ultimate panacea for cancer by correcting the fundamental genetic errors in tumors. The engineering of nanoscale inorganic carriers of cancer therapeutics has shown promising results in the efficacious and safe delivery of nucleic acids to treat oncological diseases in small‐animal models. When these nanocarriers are used for co‐delivery of gene therapeutics along with auxiliary treatments, the synergistic combination of therapies often leads to an amplified health benefit. In this review, an overview of the inorganic nanomaterials developed for combinatorial therapies of gene and other treatment modalities is presented. First, the main principles of using nucleic acids as therapeutics, inorganic nanocarriers for medical applications and delivery of gene/drug payloads are introduced. Next, the utility of recently developed inorganic nanomaterials in different combinations of gene therapy with each of chemo, immune, hyperthermal, and radio therapy is examined. Finally, current challenges in the clinical translation of inorganic nanomaterial‐mediated therapies are presented and outlooks for the field are provided. The principles of therapeutic nucleic acids, inorganic nanocarriers, and delivery of gene/drug payloads are introduced. The utilities of recently developed inorganic nanomaterials in different combinations of gene therapy with each of chemo, immune, hyperthermal, and radio therapy are discussed. Finally, current challenges facing inorganic nanomaterial‐mediated combinatorial therapies are reviewed and outlooks in the field are provided.ViewShow abstractConformational and Dynamic Properties of Short DNA Minicircles in Aqueous Solution from Atomistic Molecular Dynamics SimulationsArticleJul 2020MACROMOLECULES Terpsichori S. Alexiou Panagiotis V. Alatas Dimitrios Tsalikis Vlasis G MavrantzasViewShow moreRoles of Topoisomerases in Maintaining Steady-state DNA Supercoiling in Escherichia coliArticleFull-text availableMar 2000J BIOL CHEME Lynn ZechiedrichDNA supercoiling is essential for bacterial cell survival. We demonstrated that DNA topoisomerase IV, acting in concert withtopoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli. Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (ς) of −0.015at an initial rate of 0.8 links min−1. Topoisomerase I relaxed DNA at a faster rate, 5 links min−1, but only to a ς of −0.05. Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately thesame level as in a mutant lacking topoisomerase I activity (to ς = −0.08). The role of topoisomerase IV was revealed by twofunctional assays. Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled(ς = −0.09), greatly stimulating transcription from the supercoiling sensitive leu-500promoter and increasing the number of supercoils trapped by λ integrase site-specific recombination.ViewShow abstractEnzymatic Synthesis of Deoxyribonucleic AcidArticleFull-text availableFeb 1962J BIOL CHEMH. Vasken AposhianA KornbergViewA guide to choosing fluorescent proteinsArticleJan 2005NAT METHODSN.C. ShanerP.A. SteinbachR.Y. TsienThe recent explosion in the diversity of available fluorescent proteins (FPs) promises a wide variety of new tools for biological imaging. With no unified standard for assessing these tools, however, a researcher is faced with difficult questions. Which FPs are best for general use? Which are the brightest? What additional factors determine which are best for a given experiment? Although in many cases, a trial-and-error approach may still be necessary in determining the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.ViewShow abstractMinicircle DNA vectors devoid of bacterial DNA result in persistent and high level transgene expression in vivoArticleJan 2003 Zhiying ChenC.Y. HeA Ehrhardt Mark A KayViewSplit ubiquitin as a sensor of protein interactions in vivoArticleOct 1994Nils Johnsson Alexander VarshavskyWe describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-terminal fragment of ubiquitin (C-ub) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (N-ub) is also expressed in the same cell. This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered N-ub. However, if C-ub and the altered N-ub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C-ub is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions. This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.ViewShow abstractLMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1ArticleOct 2003SCIENCES. Hacein-Bey-AbinaWe have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as γ chain (γc) deficiency]in 9 out of 10 patients by retrovirus-mediated γc gene transfer into autologous CD34 bone marrow cells. However, almost 3years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with γδ+ or αβ+ T cell receptors)has occurred in the two youngest patients. Both patients clones showed retrovirus vector integration in proximity to theLMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, mostlikely driven by retrovirus enhancer activity on the LMO2 gene promoter.ViewShow abstractIdentification par Complémentation in vitro et Purification d un Segment Peptidique de la β-Galactosidase d Escherichia coliArticleJul 1965J MOL BIOLAgnes UllmannDavid PerrinF JACOBJacques MonodViewEffect of jet nebulization on DNA: Identifying the dominant degradation mechanism and mitigation methodsArticleAug 2005J AEROSOL SCI Yvonne K LentzL.R. Worden Thomas J Anchordoquy Corinne S LengsfeldJet nebulization is a common technique by which gene therapies can be delivered to the lungs, however current research has shown a reduction in activity and effectiveness of these treatments when not complexed with cationic agents. To date, a systematic investigation of chemical and mechanical degradation pathways has yet to be undertaken to identify the proximate cause of DNA instability. The purpose of this paper is to identify the dominant chemical and/or mechanical mechanism(s) by which DNA degrades when subjected to jet nebulization. Investigation of multiple degradation pathways revealed hydrodynamic shear to be primarily responsible for the observed loss in DNA integrity. Complexation of DNA with cationic agents polyethyleneimine or poly-L-lysine to DNA was sufficient to overcome these mechanical forces via a reduction in applied hydrodynamic forces. (C) 2004 Elsevier Ltd. All rights reserved.ViewShow abstractFlow dichroism of deoxyribonucleic acid solutionsArticleApr 1968J. Pept. Sci.Chong Sung LeeNorman DavidsonThe flow dichroism of dilute DNA solutions (A260 ≈ 0.1) has been studied in a Couette-type apparatus with the outer cylinder rotating and with the light path parallel to the cylinder axis. Shear gradients in the range of 5–160 sec.−1 were studied. The DNA samples were whole, \"half,” and \"quarter” molecules of T4 bacteriophage DNA, and linear and circular λb2b5c, DNA. For the linear molecules, the fractional flow dichroism is a linear function of molecular weight. The dichroism for linear λ DNA is about 1.8 that of the circular molecule. For a given DNA, the dichroism is an approximately linear function of shear gradient, but with a slight upward curvature at low values of G, and some trend toward saturation at larger values of G. The fractional dichroism increases as the supporting electrolyte concentration decreases.ViewShow abstractGeneration of a Library of Randomly Overlapping DNA InsertsArticleJun 2006Joseph SambrookDavid W RussellViewShow moreRecommended publicationsDiscover moreArticleStructure and Structure—Function Studies of Lipid/Plasmid DNA ComplexesFebruary 2000 · Journal of Drug TargetingAJ LinNelle L. SlackA. Ahmad[...]Cyrus R. SafinyaRecent synchrotron-based X-ray diffraction studies have enabled us to comprehensively solve the self-assembled structures in mixtures of cationic liposomes (CLs) complexed with linear lambda-DNA. In one case the CL-DNA complexes were found to consist of a higher ordered multilamellar structure (labeled L(alpha)C with DNA sandwiched between cationic bilayer membranes. The membrane charge density ... [Show full abstract] is found to control the DNA interaxial spacing with high densities leading to high DNA compaction between lipid bilayers. A second self-assembled structure (labeled H(II)C) consists of linear DNA strands coated by cationic lipid monolayers and arranged on a 2D hexagonal lattice. In this paper we report on a combined X-ray diffraction and optical microscopy study of CLs complexed with functional supercoiled plasmid DNA. We describe the self-assembled structures in cell culture medium for both a high transfectant complex (DOTAP/DOPE, phiDOPE = 0.72) and a low transfectant complex (DOTAP/DOPC, (phiDOPC = 0.72). Fluorescence optica microscopy shows two distinct interactions between these two types of complexes and mouse fibroblast L-cells, demonstrating the existence of a correlation between structure and transfection efficiency.Read moreArticleChemInform Abstract: Rational Design of Smart Supramolecular Assemblies for Gene Delivery: Chemical...November 2011 · Chemical Society Reviews Kanjiro MiyataNobuhiro Nishiyama Kazunori KataokaPolymeric materials have been extensively developed as a delivery vehicle for nucleic acids over the past two decades. Many previous studies have demonstrated that synthetic delivery vehicles can be highly functionalized by chemical approaches to overcome biological barriers in nucleic acid delivery, similar to viruses. Based on our current knowledge, this tutorial review describes rational ... [Show full abstract] strategies in the design of polymeric materials to achieve construction of the versatile vehicles, that is artificial viruses , for successful gene therapy, especially focusing on the chemical structures with the minimal adverse effects.Read moreArticleFull-text availableA new developing class of gene delivery Messenger RNA-based therapeuticsOctober 2017 · Biomaterials Science Zhao Meng Jonathan O Keeffe AhernJing Lyu[...] Wenxin WangGene therapy has long been held as having the potential to become a front line treatment of various genetic disorders. However, the direct delivery of nucleic acids to correct a genetic disorder has numerous limitations owing to the inability of naked nucleic acids (DNA and RNA) to traverse the cell membrane. Recently, messenger RNA (Mrna) based delivery has become a more attractive alternative ... [Show full abstract] to DNA due to the relatively easier transfection process, higher efficiency and safety profile. As with all gene therapy, the central challenge remains the efficient delivery of nucleic acids intracellularly. This review presents the recent progress in mRNA delivery, focusing on comparing the advantages and limitations of non-viral based delivery vectors.View full-textArticleChallenges of turning nucleic acids into therapeuticsDecember 2000 · Advanced Drug Delivery Reviews Ram I MahatoRead moreDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? 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