development of renal targeted vectors through combined in vivo phage display and capsid engineering of adenoviral fibers from serotype 19p

© The American Society of Gene Therapy original article Development of Renal-targeted Vectors Through Combined In Vivo Phage Display and Capsid Engineering of Adenoviral Fibers From Serotype 19p Laura Denby1, Lorraine M Work1, Dan J Von Seggern2, Eugene Wu3, John H McVey4, Stuart A Nicklin1 and Andrew H Baker1 British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK; 2Department of Immunology, Scripps Research Institute, La Jolla, California, USA; 3Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA; Haemostasis and Thrombosis, Medical Research Council Clinical Sciences Centre, Imperial College London, London, UK The potential efficacy of gene delivery is dictated by the infectivity profile of existing vectors, which is often restrictive In order to target cells and organs for which no efficient vector is currently available, a promising approach would be to engineer vectors with novel transduction profiles Applications that involve injecting adenovirus (Ad) vectors into the bloodstream require that native tropism for the liver be removed, and that targeting moieties be engineered into the capsid We previously reported that pseudotyping the Ad serotype fiber for that of Ad19p results in reduced hepatic transduction In this study we show that this may be caused, at least in part, by a reduction in the capacity of the Ad19p-based virus to bind blood coagulation factors It is therefore a potential candidate for vector retargeting, focusing on the kidney as a therapeutic target We used in vivo phage display in rats, and identified peptides HTTHREP and HITSLLS that homed to the kidneys following intravenous injection We engineered the HI loop of Ad19p to accommodate peptide insertions and clones Intravenous delivery of each peptide-modified virus resulted in selective renal targeting, with HTTHREP and HITSLLS-­targeted viruses selectively transducing tubular epithelium and glomeruli, respectively Our study has important implications for the use of genetic engineering of Ad fibers to produce targeted gene delivery vectors Received 15 December 2006; accepted 25 April 2007; published online June 2007 doi:10.1038/sj.mt.6300214 Introduction Gene delivery is limited by the ability of available vector systems (either viral or non-viral) to deliver sufficient levels of therapeutic transgenes to target cells and tissues in vivo to provide an efficacious phenotype For strategies based on the commonly used serotype 5,1 intravenous administration of Ad vectors results in gene delivery that is restricted only to defined cell types such as liver hepatocytes Access to local tissue can enable tissue ­transduction to be carried out, but access to therapeutically relevant targets is often impossible or impractical In this context, there is a need to develop targeting strategies to enable gene delivery to these sites in vivo Such strategies have evolved rapidly in recent years and include utilization of alternate vector serotypes as well as methodologies to alter the infectivity of existing vectors, including peptides and antibody targeting systems In many of these strategies, however, the required route of delivery is via the bloodstream with “homing” of the vector to the target tissue Alternative serotypes of some commonly used vectors have been isolated, and many possess alternative tropism on account of differences in cell tethering and entry mechanisms, thereby enabling targeting to defined tissues For example, adeno-associated virus serotypes (AAV-6) and (AAV-9) show significant levels of delivery of genes to skeletal and cardiac muscle following intravenous injection, when compared with AAV-2, thereby potentially accelerating the development of gene therapeutics for skeletal and cardiac muscle-related diseases.2–5 In Ad-based vectors for which a large number of alternate serotypes are available, altered cell infectivity can be achieved For example, Ad vectors engineered with many subgroup B fibers target CD46, thereby altering infection at the level of cell surface binding.6,7 For Ad, this has been very successful for localized gene delivery8 but less efficient when delivered via the bloodstream The fiber protein exposed on the surface of the adenoviral capsid is the main determinant of tropism, and has been the predominant target of retargeting strategies (reviewed in Ref 9) In the case of Ad5, extensive mutagenesis of the fiber has resulted in reduced liver transgene expression but has not enabled vector retargeting.10–12 This could be partly explained by the fact that there are still some uncertainties about the extent to which receptors in the liver are utilized by Ad5 vectors in vivo Recently there have been reports of the important role played by blood serum proteins, including coagulation factors IX (FIX) and X (FX), in driving hepatic delivery of Ad5 through heparan sulphate proteoglycans (HSPGs) and/or low-density lipoprotein receptorrelated protein.13,14 In view of all these findings, the retargeting of Ad vectors based on serotype will likely require elimination of coagulation factor binding Because coagulation factor binding Correspondence: Andrew H Baker, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow G12 8TA, UK E-mail: ab11f@clinmed.gla.ac.uk Molecular Therapy vol 15 no 9, 1647–1654 sept 2007 1647 © The American Society of Gene Therapy Engineering Renal-selective Gene Therapy Vectors Results Binding of coagulation factors and resulting cell infectivity is reduced with Ad19p as compared to Ad5 We previously reported that replacing the Ad serotype fiber with that of Ad19p results in reduced hepatic transduction.15 Recently it has been found that an important mechanism for hepatic sequestration of Ad vectors is the binding of blood coagulation factors, such as FIX and FX, to the capsid so as to “bridge” the virus to HSPGs and/or low density lipoprotein ­receptor-related protein in the liver.13,14 We first determined whether physiological concentrations of FIX or FX exerted any influence on Ad19p-mediated transduction, and compared the results with Ad5 (using a wildtype fiber, Ad5 and a coxsackievirus and Ad receptor bindingdeleted capsid, AdKO123) using human hepatoma cells (HepG2) In conformity with the results of our previous study,15 HepG2 cell transduction was lower with Ad19p than with Ad5 (Figure 1a) As expected,14 both Ad5 and AdKO1 transductions were enhanced by both FIX and FX Ad19p-mediated transduction was also s­ignificantly enhanced by FIX and FX, but to substantially lower 1648 a 16 RLU/mg protein (× 10 ) 14 12 10 + FIX + FX 140 b 160 àg -galactosidase (ì 10 )/mg protein Ad5 + FIX + FX + FIX + FX AdKOI Ad19p CHO-KI CHO-pgsA745 120 100 80 60 40 20 − IX FX − FIX FX + +F + + − Ad5 c IX FX − FIX FX + + + +F Ad19p-Eco47 Virus injection end Resp diff (RU) start �RU �RU Ad5 Ad19p-Eco47 Time (s) d 100 75 �RU is probably mediated through the fiber,13 an alternative approach would be to identify serotypes that show reduced coagulation factor binding We previously reported that the fiber of serotype 19p, a subgroup D virus not associated with any known human disease, exhibits severely reduced liver tropism when delivered intravenously into rats, and this was in the absence of enhanced targeting to any other organ.15 This vector therefore represents a potential platform on which to build a targeting system for delivery of genes to non-hepatic tissue As stated earlier, peptides9 and antibodies16 have shown potential for in vivo targeting of Ad following intravascular injection, and appropriate targeting sequences can be identified by techniques including in vivo phage display The flexibility of phage display allows entire peptide repertoires to be screened in vitro, ex vivo, or in vivo in order to isolate highly efficient and selective targeting sequences.17 Importantly, studies have demonstrated that it is possible to identify small peptide motifs that target the vasculature of defined tissues and organs following intravenous administration of phage libraries.17–20 The translation of this technology to the delivery of bioactive therapeutics, using peptides isolated by phage display, has potential clinical utility, and this has already been proven in a limited range of applications For example, intravenous administration of peptides isolated for specifically targeting tumors or white fat, when coupled to drugs and pro-apoptotic peptides, resulted in tumor regression or fat ablation, respectively.18,21,22 Translation of this technology into efficient and selective in vivo gene delivery by viral vector systems has been somewhat slower to emerge, because this involves genetic engineering of complex viral capsid proteins We have therefore utilized Ad5 pseudotyped with the Ad19p fiber in order to develop targeted viral gene therapy vectors by intravenous administration, by combining in vivo phage display with the genetic engineering of the HI loop of Ad serotype 19p fibers We have focused on the kidney as a therapeutic target because it is fundamentally important in a range of diseases and there is a clear absence of suitable vector systems for non-invasive targeting Ad5 Ad19p-Eco47 50 25 0.00 0.25 0.50 0.75 1.00 11 Virus titre (× 10 ) 1.25 Figure 1  Assessment of coagulation factor binding by modified viruses (a) HepG2 cells were distributed in the plates 24 hours prior to infection Plated cells were washed in phosphate-buffered saline (PBS) and 50 µl of serum free media with physiological levels (1 IU/ml) of either factor IX (FIX) or FX added This was followed by infection with the appropriate Ad at 10,000 virus particles (VP)/cell Cells were incubated for hours at 37 °C before being washed and the media replaced Cells were incubated for 72 hours before quantification of transgene expression *P < 0.05 versus transduction in the absence of clotting factors (b) CHO-K1 (wild-type) and CHO-pgsA745 (heparan sulphate proteoglycan-deficient)24 cells were used for elucidating the effects and mechanisms of coagulation factors on Ad19p-modified vector transduction Plated cells were washed in PBS and 50 µl of serum free media with physiological levels (1 IU/ml) of either FIX or FX added This was followed by infection with 20,000 VP/cell of the appropriate Ad Cells were incubated for hours at 37 °C before being washed and the media replaced Cells were incubated for 48 hours before quantification of transgene expression *P < 0.05 versus transduction in the absence of clotting factors (c, d) Ad5 or Ad19p vectors at various concentrations (×1011 VP/ml) were perfused over FX immobilised onto a CM-5 sensor chip in 50 mmol/l Tris pH 7.4; 150 mmol/l NaCl; 5 mmol/l CaCl2; 0.005% Tween 20 at a flow rate of 20 μl/min at 25 °C Depicted are sensorgrams of c FX (showing specific binding) of Ad5 and, to a lesser extent, Ad19p-Eco47 (d) The steady state change (δRU) in respiratory unit (RU) on binding of the virus to the FX was plotted against virus concentration CHO, Chinese hamster ovary; RLU, relative light units www.moleculartherapy.org vol 15 no sept 2007 © The American Society of Gene Therapy Engineering Renal-selective Gene Therapy Vectors levels than with AdKO1 and Ad5 (Figure 1a) This suggests that Ad19p may show reduced binding to coagulation factors To investigate this further we utilized two approaches, one involving Chinese hamster ovary (CHO) cells and the other involving surface plasmon resonance (SPR) First, wild-type CHO-K1 cells and HSPG polymerisation-­deficient CHO cells (CHO-pgsA745)24 were transduced with Ad5 and Ad19p in the presence or absence of FIX and FX Basal levels of transduction for both Ad5 and Ad19p were low, likely reflective of lack of primary receptor availability on CHO cells (Figure 1b) Ad5 transduction was significantly enhanced in the presence of both FIX and FX (Figure 1b), an effect that was ablated in CHO-pgsA745 cells, thereby demonstrating that coagulation factor-mediated cell transduction relies on HSPG targeting.13,14 In the case of Ad19p, FX (but not FIX) showed enhanced transduction in CHO-K1 cells but, in agreement with the data for HepG2 cells (Figure 1a) this was to a level lower than with Ad5 (Figure 1b) Again, this effect was deficient in CHO-pgsA745, thereby demonstrating that binding of the Ad19p:FX complex to the cell surface occurs through HSPG (Figure 1b) Next, in order to examine the direct interaction of the Ad5 and Ad19p viruses with FX, we carried out SPR analysis FX was covalently coupled to biosensor chips and the virus (at varying concentrations) was injected Both viruses showed specific binding to FX, but there was a substantially weaker interaction in the case of Ad19p (Figure 1c and d), and this interaction was calcium-dependent, consistent with the characteristics associated with binding of Ad5-based vectors.14 When quantified, Ad19p showed substantially reduced binding to FX in comparison with Ad5 (Figure 1c and d), consistent with the HepG2 and CHO cell transduction data (Figure 1a and b) This demonstrates that viruses with fibers derived from Ad19p show lower levels of binding and sensitivity to the coagulation factor pathway than Ad5 does This, at least in part, may be the underlying cause of the reduced liver tropism of Ad19p as compared to Ad5,15 and suggests that Ad19p fibers have a potential role to play in vector targeting strategies with intravascular injection routes a Identification of renal homing peptides by in vivo phage display Using Wistar Kyoto (WKY) rats, we carried out three rounds of in vivo phage display to identify peptides that had the ability to target the kidneys following intravenous injection (Figure 2a) Twelve- to thirteen-week-old WKY rats (n = rats/phage display round) were infused with × 1011 plaque-forming units (PFU) of PhD 7mer library in round 1, and with × 1011 PFU of amplified phage from the kidney for rounds and (Figure 2a) Phage were recovered, titered and normalized per gram of tissue By the third round, we observed selective enrichment of the phage display library for the kidney in comparison with non-target organs (Figure 2b) We then performed DNA sequence analysis of phage in the kidneys (>200 individual phage for rounds and 3) (Table 1), and from non-target organs (heart, brain, and lungs (>100 individual phage))—Table  in order to identify the encoded peptides It was clear that there were some peptides found only in one organ, while others such as APASLYN were found in all tissues This highlights the fact that phage display has the potential to identify peptides on the basis of either ubiquitous or tissue-selective markers From the sequencing analysis, the peptides HTTHREP (HTT) and HITSLLS (HIT) were selected for the experiments, as they were found to be exclusively selective for the kidneys (Tables 1 and 2) In order to assess the kidney targeting capacity of each peptide, we injected animals with a high titer stock of each individual phage We utilized a pre-dosing regimen wherein animals were injected with control (not expressing a peptide) phage 5 ­minutes prior to injection of the candidate peptide-expressing phage (Figure 3a), as we have described previously.25 Pre-dosing was b Phage IV infusion circulation, saline perfusion Recycle Kidney retrieval Titer restricted libary Sequence Phage recovery (% of liver) 1st RD 2nd RD 3rd RD NS NS NS NS Heart Brain Kidney Lung Phage recovery and amplification Figure 2  In vivo phage display (a) Schematic representation of in vivo phage display in the rat, so as to identify renal targeting peptides Recovered and amplified phage from the kidneys minutes after injection were subjected to a total of three rounds of in vivo phage display, followed by sequence analysis after round and (b) Phage recoveries over sequential rounds of phage display *indicates P < 0.05, NS, nonsignificant IV, intravenous Table 1 Consensus sequences identified for the kidney following three rounds of in vivo phage display Linear 7mer library n/total phage sequenced Round AYPYPHT 2/214 Rd APASLYN 4/214 Rd HITSLLS 6/214 Rd and HTTHREP 5/214 Rd NNATLG 2/214 Rd NQDVPLF 3/214 Rd NSLMRPA 2/214 Rd QGFGSPL 2/214 Rd THHPHQK 2/214 Rd Table 2  Phage isolated from non-target organs were sequenced following the third round of in vivo phage display TLARPSV 2/214 Rd Heart Brain Lung TTNGYI 3/214 Rd APASLYN (4.88%) APASLYN (2.27%) APASLYN (2.27%) WDPPLRL 2/214 Rd HAAIHIS (4.88%) LPKNWSS (4.55%) VLTAGPW (7.695) WHRAPLP 2/214 Rd YLQAPVH (4.88%) LVSQPHP (4.55%) A total of 214 phage were sequenced Molecular Therapy vol 15 no sept 2007 A total of 111 phage were sequenced 1649 © The American Society of Gene Therapy Engineering Renal-selective Gene Therapy Vectors b Control NS c b HI loop NS 10 × 10 11 × 10 11 × 10 NS NS NS Liver Kidney Lung Heart 10 HTT NS Ad19p Ad19p-Eco47 10 10 10 10 10 10 10 Kidney Liver 30 25 20 15 10 5 20 Ad19p e d kd 20 Ad19p-Eco47 Ad19p Ad19p parental -Eco47 HTT HIT 50 35 30 10.84 12 Phage recovery (expressed as a % of liver) 40 35 c 10 Ad19p-HTT Ad19p-HIT 1.17 0.17 Control HIT HTT Figure 3  Analysis of selected peptide-expressing phage (a) Demonstration of saturation kinetics of M13 phage in the liver, kidney, lung, and heart at increasing doses *indicates P < 0.05 versus control phage (b) Phage recoveries from kidney and liver (plaque forming units (PFU)/g tissue) from animals infused with × 1010 PFU per animal of either control phage or targeted phage (as indicated) following 2 × 1011 PFU per animal control phage to saturate non-specific binding *P < 0.05 versus control phage (c) Phage recoveries expressed as a percentage of phage in the liver Individual values shown above each bar NS, non significant 1650 a HIT Phage recovery (PFU/g tissue) % Of input −3 (phage recovered/input dose) (× 10 ) a Ad5 as a further control) to determine the extent and selectivity of renal targeting Immunohistochemical analysis of kidneys from Ad5- or Ad19p-Eco47-injected rats showed no transgene product (β-galactosidase) (Figure 5) In contrast, both peptidemodified vectors showed readily detectable levels of transgene in the kidneys, to a ­ significantly higher level than that seen in the control virus- or Ad5-infused animals (Figure 5) The different ­peptide-modified vectors produced different cellular distri­­ bution patterns in the kidneys Ad19p-HIT showed extensive �-gal (RLU × 10 )/mg protein carried out in order to pre-saturate non-specific phage binding sites in the liver, so as to analyze peptide-mediated targeting without the confounding factor of reticulo-endothelial entrapment of the phage particle The biodistribution of each phage following the pre-dosing regimen (Figure 3b) showed a significantly elevated phage accumulation for HIT and HTT in the kidney as compared to the control phage, with no significant changes seen in the liver When the recovery of either HITSLLS, HTTHREP or control phage from the kidney was expressed as a percentage of the liver, it was approximately tenfold higher in HITSLLS infused animals and approximately 100-fold higher in HTTHREP infused animals, in comparison with control phage (Figure 3c) In order to produce targeted gene therapy vectors based on Ad19p, we genetically modified the fiber gene from Ad19p to accommodate peptide insertions, and inserted oligonucleotides encoding each peptide individually into the HI loop (a site that has been successful for targeting Ad5 fibers using peptides26,27) Alignment of the Ad5 and 19p fiber nucleotide sequences showed that the HI loop of 19p was considerably smaller than Ad5 and did not contain a suitable restriction site for insertion of oligonucleotides encoding each targeting peptide We used a polymerase chain reaction (PCR) strategy (see Materials and Methods and Figure 4a) to insert an Eco47III restriction site between amino acids 331 and 332 in order to facilitate the insertion of oligonucleotides for renal-targeting peptides (see Materials and Methods) The resulting virus (Ad19p-Eco47) (Figure 4b) was compared with parental Ad19p (non-modified fiber) to confirm there were no differences in in vitro infectivity (Figure 4c) Subsequently we cloned oligonucleotides encoding each candidate renal targeting peptide into the Ad19p-Eco47 fiber-expressing plasmid and produced peptide-modified vectors (model shown in Figure 4d) Levels of virus production (>1 × 1012 viral particles/ml) and fiber levels were equivalent to those observed with non-modified fibers, as assessed by Western blot analysis (Figure 4e) Having derived all peptides by in vivo phage display, and given that the critical aspect of targeting is expression of the encoded transgene in vivo, we proceeded to inject rats with each virus We killed animals days after injection of each virus (and Figure 4 Engineering of Ad19p fibers for targeting peptide insertion (a) Schematic illustrating the protocol followed for producing the modified vectors (refer to Materials and Methods) (b) Model of the predicted structure of the Ad19p fiber with and without insertion of the restriction site to allow cloning (c) In vitro comparison of Ad19p and Ad19p-Eco47 Rat glomerular endothelial cells were infected with increasing doses of either virus for hours at 37 ºC They were then washed and the media was replaced Seventy-two hours after infection, cells were harvested and β-galactosidase was measured and normalized to protein (d) Model of the predicted structure of the Ad19p fiber with each peptide inserted into the HI loop (e) Western blot of fiber monomer Ten µg of viral protein was loaded, and the membrane was probed with the anti-fiber antibody 4D2 (Neomarkers, Fremont, CA) at 1:1,000 Ad, adenovirus; PCR, polymerase chain reaction; RLU, relative light units www.moleculartherapy.org vol 15 no sept 2007 © The American Society of Gene Therapy a Low power Engineering Renal-selective Gene Therapy Vectors High power a Ad19p Eco47 Ad19p HIT Ad19p HTT Liver Ad5 Ad5 Spleen Ad19p-Eco47 Ad19p-Eco47 Heart b 90 80 Ad19p-HIT Ad19p-HIT Ad19p-HTT Ad19p-HTT % Positive cells 70 b 60 50 40 30 20 50 10 40 30 Ad5 NS 20 10 PBS Ad5 Ad19p-Eco47 Ad19p-HIT Ad19p-HTT Figure 5  Analysis of kidney targeting in vivo Eight-week old male Wistar Kyoto rats were infused with 3.5 × 1011 virus particles per rat of each modified vector or phosphate-buffered saline (PBS) and killed days later (a) Immunohistochemistry performed in kidney sections Black staining indicates β-galactosidase activity Representative sections are shown (n = rats per group) Scale bar = 100 µm (b) Quantitative analysis of transgene-expressing cells *P < 0.05 versus Ad19p-Eco47injected rats and Ad5-injected rats NS, not significant Ad, adenovirus l­ ocalization within the glomerulus with little or no staining in other cell types (Figure 5) Ad19p-HTT showed substantial and selective staining within the epithelial cells of the tubules (Figure 5) Immunohistochemical analysis revealed no transgene expression in the liver for any Ad19p-based virus (Figure 6) Further analysis of non-target organs including spleen, heart (Figure 6a), lungs, and brain (not shown) also failed to detect transgene expression In our previous study on Ad19p liver targeting ­ following intravascular injection, we used a pre-dosing regimen to assess hepatocyte transduction in the absence of Kupffer cell clearance.15 We repeated that strategy here and assessed targeting of Ad5, Ad19p-Eco47, and both peptidemodified vectors Levels of β-galactosidase-positive hepatocytes were: for Ad5, (83 ± 1%); for Ad19p-Eco47, (9 ± 1%); for Ad19pHIT, (4 ± 1%); and for Ad19p-HTT, (9 ± 1%) (Figure 6b) That is, liver targeting of modified Ad19p vectors was not altered as compared to Ad19p control virus, whether in the presence or absence of Kupffer cell clearance Hence, each peptide-modified vector ­produced ­efficient and selective transgene expression in the kidneys following intravenous administration Finally, we harvested organs hour after injection of the same dose of virus in order to assess early virion accumulation in the liver and kidneys As expected, virion levels in the liver were Molecular Therapy vol 15 no sept 2007 Ad19p-Eco47 Ad19p-HIT Ad19p-HTT Figure 6  Analysis of non-renal tissue for reporter gene activity (a) Eight-week old male Wistar Kyoto rats were infused with 3.5 × 1011 virus particles per rat of each modified vector or phosphate-buffered saline and killed days later Immunohistochemistry was performed on the liver, spleen, and heart Black staining indicates β-galactosidase activity Representative sections are shown (n = rats per group) Scale bar = 100 µm (b) Quantitative analysis of transgene-expressing cells from liver sections of animals infused using a pre-dose strategy Ad, adenovirus 10,000 Ad19p Eco47 Ad19p HIT 1,000 Virus particles/ng DNA % Positive cells 60 Ad19p HTT 100 10 0.1 Kidney Liver Figure 7  Assessment of early particle delivery to liver and kidney Eight-week old male Wistar Kyoto rats were infused with 3.5 × 1011 virus particles per rat of each modified vector or phosphate-buffered saline and killed following perfusion at hour after injection Virion quantification was carried out by means of Taqman Data Analysis software (Applied Biosystems), using SYBR green DNA was extracted from the kidney and 200 ng total DNA was amplified using LacZ primers, and the products were quantified using Taqman *P < 0.05 versus Ad19p-Eco47 Ad, adenovirus ­relatively high but essentially equivalent for Ad19p-Eco47, Ad19pHIT and Ad19p-HTT, likely as a result of profound Kupffer cell interactions However, although levels in the kidney at hour after injection were far lower than in the liver, significant increases were nevertheless observed with both peptide-modified Ad19p vectors (Figure 7) This highlights the potential of the engineered Ad19p 1651 Engineering Renal-selective Gene Therapy Vectors system for mediating selective kidney transduction even in the presence of rapid Kupffer cell uptake It also illustrates that strategies to ablate the Kupffer cell interaction of Ad19p-based vectors may further enhance targeting potential Discussion In this study we rationally combined in vivo phage display with engineering of the Ad19p fiber to accommodate incorporated peptides within the constraints of the fiber’s minimal HI loop We showed that Ad19p-based vectors have lower coagulation factor binding and sensitivity to this pathway when compared with Ad5, thus contributing to the reduced hepatic tropism of the vectors Furthermore, we developed in vivo phage display to identify renal targeting peptides and demonstrated the incorporation of these peptides into Ad19p pseudotyped vectors In vivo we showed selective targeting to the kidney Of particular importance is the finding that specific cell types in the kidney are targeted by different peptides, because this suggests a broadly applicable approach to targeting not only selected organs but also individual cell types within each organ Retargeting of Ad vectors for selective gene therapy via the intravenous route has been widely attempted but has been hampered by a number of issues including complex interplay in vivo involving red and white blood cells,10,28 platelets29 and serum coagulation factors,13,14 and complement proteins,30,31 all of which may contribute to the tropism of Ad and also to the host innate immune responses Although a variety of capsid modifications and peptide insertions have been utilized to generate convincing data showing enhanced and/or selective gene delivery in vitro or locally,26,27 there have been no studies to date that have convincingly retargeted genetically-engineered Ads following intravenous injection We used in vitro cell gene transfer experiments and SPR to assess direct FX:virus binding as well as to show that viruses based on Ad19p showed reduced sensitivity to coagulation factor binding when compared with Ad5 The binding of FIX to Ad5 has been shown to occur within the fiber structure.32 In view of the fact that Ad5 infection has been shown to be heparin sensitive, the KKTK motif in the fiber shaft has been suggested as a putative motif that mediates interaction with HSPGs,33,34 and mutation of the KKTK motif reduces liver transduction of Ad5 in vivo.10–12 However, an Ad5 vector with a mutated KKTK motif binds coagulation FX as effectively as does non-modified virus, thereby demonstrating that this is not the site of direct interaction.35 It is important to determine the interaction locale in order to clarify and interpret the lowered binding capacity observed with Ad19p fibers In parallel with this, we have recently shown that many other Ad5-based vectors with fibers from subgroup D bind coagulation factors,36 leading to enhanced transduction of cells in vitro In future studies, therefore, it will be critically important to ascertain affinity constants for different interactions between Ad vectors with alternative fibers Although Ad5 viruses have been targeted using different sites within capsid proteins, including the fiber HI loop, penton, hexon, and pIX,9 we concentrated on engineering of the Ad19p HI loop, because SWISS-MODEL analysis suggested that peptide exposure at the fiber surface would be optimal Although the HI loop of Ad19 is much smaller than that of Ad5, we were able to 1652 © The American Society of Gene Therapy document efficient virus production with each of the peptidemodified viruses created The incorporation of relatively small peptides into this site was shown not to hinder virus assembly propagation The principal thrust of our strategy was to modify a virus fiber that naturally shows poor liver tropism and combine this with peptide engineering Depending on the extent of modification in the Ad19p fiber HI loop that can be tolerated without hindering virus production, it may be possible to use engineered peptides in tandem or, indeed, direct larger targeting peptides into this locale However, targeting with 7-mer peptides isolated by phage display has been shown to be efficient for renal gene delivery It may be possible to improve targeting even further by strategies to block Kupffer cell interactions This would (potentially) enhance the bioavailability of peptide-modified Ad for the kidney While the receptor(s) for Ad19p remains unknown, we have shown previously that the Ad19p fiber supports the infection of rat endothelial cells15 as well as rat-derived smooth muscle cells (data not shown), thereby suggesting that the receptor(s) utilized by Ad19p is present on rat tissue This allows the systematic analysis of vector retargeting strategies under conditions in which the primary receptor for the parental fiber is present Ad19p can also infect human saphenous vein endothelial cells and smooth muscle cells15 (as well as the A549 cell line37) This would suggest that the primary receptor targeted by Ad19p has a similar pattern of expression in rats and humans, although further work is required to elucidate these similarities fully To date, renal gene therapy has been achieved by applying the vector locally.38,39 Intravenous delivery has been feasible only by using an alternative, easily accessible tissue or organ such as liver, muscle, or skin as a “transgene-secreting factory”.40–43 While this is useful for analyzing individual transgenes in pre-clinical settings, its use is limited to secreted transgene products only The vectors we used in our study will allow high-level transgene expression locally, but via the non-invasive intravenous route Additionally, strategies for targeting individual cells within the kidney can be adopted, with specific peptide-modified vectors to target the glomerulus or tubular epithelium, as required Furthermore, these vectors are especially important in studies on rats, and represent unique molecular tools, given that transgenic strategies are by no means routine In the process of our study, with all aspects taken together, we have rationally designed a new generation of adenoviral ­vectors that achieve efficient transgene expression in the kidneys following intravenous delivery We further demonstrate targeting of defined cell types within this organ This has important implications for the future design of genetically engineered Ad vectors as well as for the development of renal gene therapy, an area of research hitherto hampered by a lack of suitable vectors Materials and Methods All in vivo work was carried out under the UK Home Office regulation Animals (Scientific Procedures) Act 1986 Virus preparation and purification Stocks of recombinant pseudotyped Ads expressing the modified fibers were generated by transfection of 293T cells (American Type Culture Collection, Manassas, VA) with the modified plasmids followed by superinfection with a fiber-deleted Ad5 vector.44,45 Briefly, 293T cells were transfected with the appropriate fiber-expressing www.moleculartherapy.org vol 15 no sept 2007 © The American Society of Gene Therapy plasmid Sixteen hours later cells were superinfected with an E1, E3, fiberdeleted rAd5 (Ad5ΔF) (β-galactosidase) at 2,000 virus particles (VP)/cell Virus particles were purified by CsCl ultracentrifugation and dialyzed into 10 mmol/l Tris (pH 8.1), 150 mmol/l NaCl and 10% glycerol Virus particles were quantified by protein assay against bovine serum albumin standards according to the conversion: 1 μg protein = × 109 VP.44 Fiber expression was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot assay using monoclonal anti-fiber antibody 4D2 (Neomarkers, Fremont, CA) In vitro virus charcterisation For coagulation factor experiments, cells were distributed in a 96-well plate 24 hours prior to infection, incubated with serum-free media containing 20,000 VP/cell and 1 IU/ml of either human Factor IX or human Factor X (Haemotological Technologies, VT), left for hours at 37 °C, washed, and maintained until harvesting Forty-eight hours after infection β-galactosidase activity was quantified by plate assay using a Wallac Victor luminometer and recombinant βgalactosidase as a standard A bicinchoninic acid assay on the cell lysates was performed to determine protein concentration, and the results were expressed as relative light units/mg of protein Similarily, for transduction experiments, cells were distributed in a 96-well plate 24 hours prior to infection, incubated with fresh media containing the required multiplicity of infection (as indicated), left for hours, washed, and maintained until harvesting Seventy-two hours after infection β-galactosidase activity was quantified SPR SPR experiments were carried out with a Biacore X instrument (Biacore, Stevenage, UK) Blood coagulation FX was immobilized onto a CM5 biosensor chip according to the manufacturer’s instructions (1,926 respiratory unit of FX was immobilized) Virus in 50 mmol/l Tris pH 7.4; 150 mmol/l NaCl; 5 mmol/l CaCl2; 0.005% Tween 20 was passed over the chip at a flow rate of 20 μl/min Sensor chips were regenerated by injection of 10 mmol/l HEPES pH 7.4; 150 mmol/l NaCl; 3 mmol/l EDTA; 0.005% Tween 20 In vivo phage display The PhD Phage Display Peptide Library Kit was purchased from New England Biolabs (Hertfordshire, UK) Phage display was carried out as previously described.46 Briefly, 12–13-week-old WKY rats (n = 3 rats/phage display round) were anesthetized (halothane/O2 mixture) For round 1, × 1011 PFU of PhD library were infused into the femoral vein, and for rounds ×2 and 3, × 1011 PFU of amplified phage from the kidney were infused (Figure 2) Five minutes after infusion the animals were perfused through the heart at physiological pressure (120 mm Hg), and the organs were removed and snap frozen Phage were recovered and titered and normalized/gram of tissue Peptide sequencing Peptide-encoding DNA of phage were sequenced after rounds and Up to 96 individual plaques/rat were selected and amplified PCR was utilized for amplifying the region of the peptide insertion using sense 5′-gca att cct tta gtg gta cc-3′ and antisense 5′CCC TCA TAG TTA GCG TAA-3′ primers PCR products were seq­uenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) and analyzed on an ABI 377 automated sequencer Analysis of selected peptide-expressing phage Animals were infused with × 1011 PFU/rat of a control M13 phage lacking the LacZ α-­complementation Control phage was administered to block non-specific binding Five minutes after infusion × 1010 PFU of each individual phage (HITSLLS and HTTHREP) were infused Five minutes after infusion animals were perfused through the heart at physiological pressure (120 mm Hg), and organs removed and snap frozen.25 Phage in target and non-target organs were recovered and titered and normalized/gram of tissue Generation of peptide-modified Ad19p fibers The Ad19p fiber comple- mentary DNA in the plasmid pDV145 was constructed as previously described.15 The HI loop fragment of Ad19p was excised on the Eco065I Molecular Therapy vol 15 no sept 2007 Engineering Renal-selective Gene Therapy Vectors and XbaI sites The HI loop was modified using primers to insert a unique cloning site Eco47III Using PCR, overlapping fragments were amplified using two sets of primers The initial set of primers (i) 5′-GGA GTC GCG CAG CTT GTT GAC CAG CTC GGC-3′ and (ii) 5′-CCA ACT AAA GTC AAA TGT GAT AGA GTA TTC ACA TCC AGC GCT AGT TTC TTG GTT AAA GG-3′ (Eco47III site underlined) amplified a 1,316-­basepair fragment containing the HI loop Similarly, a second set of primers, (iii) 5′-C CAA GAA ACT AGC GCT GGA TGT GAA TAC TCT ATC ACA TTT GAC TTT AGT TGG-3′, and (iv) 5′-GGC TGG CAA CTA GAA GGC ACA GTC GAG GCT GAT CAG C-3′ (Eco47III site underlined) amplified a 190-basepair fragment The two fragments were then subjected to PCR using the flanking primers and to produce a HI loop fragment (1,435 basepairs) with an additional Eco47III site to create a HI loop sequence of NQETSAGCE (inserted amino acids underlined) The mutated HI loop fragment was excised using the Eco065I and XbaI sites and ligated into pDV145 to create pDV145-Eco47III Oligonucleotides encoding the selected peptides HTTHREP and HITSLLS were purchased from MWG (Milton Keynes, UK) and ligated into Eco47III-digested pDV145-Eco47III and sequenced to ensure correct orientation The plasmids pDV145HITSLLS and pDV145-HTTHREP were produced and used for generating Ad19p-HIT and Ad19p-HTT viruses, respectively Peptide:fiber modelling Ad19p fiber knob protein sequences with selected insertions in the HI loop were submitted to SWISS-MODEL (http://swissmodel.expasy.org/) and modeled using ExPDB templates derived from 1uxb.pdb,47 a crystal structure of Ad19p fiber knob in complex with sialic acid The bound sialic acid does not change the conformation of the fiber knob47 and was therefore ignored during modeling The models generated were visualized through Swiss-PdbViewer (http://www expasy.org/spdbv/) In vivo virus administration and analysis of virion levels and gene ­delivery In order to assess transgene expression, 8-week-old male WKY rats were infused with 3.5 × 1011 VP each Five days after infusion, the animals were killed and the organs were removed For assessing transgene expression in the absence of Kupffer cells, a pre-dose protocol was followed.15 Briefly, 8week-old male WKY rats were infused with × 1011 VP Ad null (no transgene) followed hours later by × 1010 VP of peptide-modified Ad or control virus Five days after infusion, the animals were killed and the organs were removed In order to detect transgene expression, the organs were fixed in formalin and wax-embedded Immunohistochemistry was performed on 6 μm sections, using a rabbit polyclonal anti-β-galactosidase antibody diluted 1/1,000 or matched rabbit IgG non-immune control (DAKO, Ely, UK) Detection was with biotinylated anti-rabbit secondary antibody, ABC kit and diaminobenzidine staining supplemented with nickel (all Vector Laboratories) Sections were counterstained with haematoxylin For the purpose of assessing particle delivery at an early time point after injection (1 hour) animals (n = per group) were perfused at physiological blood pressure with saline, and the organs were removed and snap frozen DNA was extracted from a defined tissue mass (Qiagen, Crawley, UK) and quantified using the Nanodrop ND-1000 (Wilmington, NC) A LacZ quantification standard curve was constructed from serial dilutions of each Ad by use of SYBR green (Applied Biosystems, Warrington, UK) with 200 nmol/l sense (5′-TAC TGT CGT CGT CCC CTC AAA-3′) and antisense (5′-TAA CAA CCC GTC GGA TTC TCC-3′) LacZ primers The total DNA was amplified, and the products were quantified using Taqman Data Analysis software (Applied Biosystems, Warrington, UK) The following reaction conditions were in use: 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particles pseudotyped with mutagenized fiber proteins show diminished infectivity of coxsackie B-adenovirus receptor-bearing cells J Virol 75: 2972–2981 46 Work, LM, Nicol, CG, Denby, L and Baker, AH (2004) In vivo biopanning: a methodological approach to identifying novel targeting ligands for delivery of biological agents to the vasculature Methods Mol Med 108: 395–413 47 Burmeister, WP, Guilligay, D, Cusack, S, Wadell, G and Arnberg, N (2004) Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites J Virol 78: 7727–7736 www.moleculartherapy.org vol 15 no sept 2007 ... for in vivo targeting of Ad following intravascular injection, and appropriate targeting sequences can be identified by techniques including in vivo phage display The flexibility of phage display. .. the Ad19p fiber in order to develop targeted viral gene therapy vectors by intravenous administration, by combining in vivo phage display with the genetic engineering of the HI loop of Ad serotype. .. targeting potential Discussion In this study we rationally combined in vivo phage display with engineering of the Ad19p fiber to accommodate incorporated peptides within the constraints of the