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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 5 minutes after injection were subjected to a total of three rounds of in vivo phage display, followed by sequence analysis after round 2 and 3. (b) Phage recoveries over sequential rounds of phage display. *indicates P < 0.05, NS, nonsignificant. IV, intravenous.
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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 (...
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Context 1
... 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 = 3 rats/phage display round) were infused with 2 × 10 11 plaque-forming units (PFU) of PhD 7mer library in round 1, and with 2 × 10 11 PFU of amplified phage from the kidney for rounds 2 and 3 ( Figure 2a). ...
Context 2
... 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 = 3 rats/phage display round) were infused with 2 × 10 11 plaque-forming units (PFU) of PhD 7mer library in round 1, and with 2 × 10 11 PFU of amplified phage from the kidney for rounds 2 and 3 ( Figure 2a). Phage were recovered, titered and normalized per gram of tissue. ...
Context 3
... 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 analy- sis of phage in the kidneys (>200 individual phage for rounds 2 found in all tissues. ...
Context 4
... Briefly, 12-13-week-old WKY rats (n = 3 rats/phage display round) were anesthetized (halothane/O 2 mix- ture). For round 1, 2 × 10 11 PFU of PhD 7 library were infused into the fem- oral vein, and for rounds ×2 and 3, 2 × 10 11 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. ...
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Citations
... One strategy to enable cellular targeting is to modify the surface molecules that interact with cellular receptors or molecules that facilitate viral entry. Modification of these viral proteins and thus a change in virus tropism has been achieved by introducing point mutations, using adapter molecules, inserting peptides or cysteine-moieties enabling coupling of the maleimide-activated ligands or pseudo-typing [42][43][44][45][46][47][48]. Different serotypes of adeno-associated viral vectors exist naturally due to variations of their capsid proteins, however, these serotypes demonstrate a preference for a tissue and not specific cellular tropism. ...
... Different serotypes of adeno-associated viral vectors exist naturally due to variations of their capsid proteins, however, these serotypes demonstrate a preference for a tissue and not specific cellular tropism. Much work on capsid engineering has been done, including using high-throughput, unbiased methods, such as directed evolution, to improve the specificity of adeno-associated vector to endothelial cells [43,47,[49][50][51]. However, this strategy is not feasible for all viral vectors, for example for lentiviral vectors a change in the viral surface protein can negatively affect viral production [52]. ...
TThe vascular endothelium serves as a semi-selective permeable barrier as a conduit for transport of fluid, solutes and various cell populations between the vessel lumen and tissues. The endothelium thus has a dynamic role in the regulation of coagulation, immune system, lipid and electrolyte transport as well as neurohumoral influences on vascular tone and end-organ injury to tissues such as the heart and kidney. Within this framework, pharmacologic strategies for heart and kidney diseases including blood pressure, glycemic control and lipid reduction provide significant risk reduction yet certain populations are at risk for substantial residual risk for disease progression and treatment resistance and often have unwanted off-target effects leaving the need for adjunct, alternative targeted therapies. Recent advances in techniques in sequencing and spatial transcriptomics have paved the way for the development of new therapies for targeting heart and kidney disease that include various gene, cell and nano-based therapies. Cell-specific endothelium-specific targeting of viral vectors will enable their use for the treatment of heart and kidney diseases with gene therapy that can avoid unwanted off-target effects, improve treatment resistance and reduce residual risk for disease progression.
... Usage of the iRGD motif further increased tumor penetration and anti-tumor effects of oncolytic adenoviral vectors [240]. Lastly, another successful strategy of adenoviral retargeting is the pseudotyping of adenovirus serotype 5 (Ad5) fiber with that of serotype Ad19p, leading to reduced transduction of liver tissue presumably by a reduced affinity for blood coagulation factors [231]. Additional insertion of phage-selected peptides HTTHREP and HITSLLS yielded selective targeting to the renal vasculature in rats [231]. ...
... Lastly, another successful strategy of adenoviral retargeting is the pseudotyping of adenovirus serotype 5 (Ad5) fiber with that of serotype Ad19p, leading to reduced transduction of liver tissue presumably by a reduced affinity for blood coagulation factors [231]. Additional insertion of phage-selected peptides HTTHREP and HITSLLS yielded selective targeting to the renal vasculature in rats [231]. ...
Forming the inner layer of the vascular system, endothelial cells (ECs) facilitate a multitude of crucial physiological processes throughout the body. Vascular ECs enable the vessel wall passage of nutrients and diffusion of oxygen from the blood into adjacent cellular structures. ECs regulate vascular tone and blood coagulation as well as adhesion and transmigration of circulating cells. The multitude of EC functions is reflected by tremendous cellular diversity. Vascular ECs can form extremely tight barriers, thereby restricting the passage of xenobiotics or immune cell invasion, whereas, in other organ systems, the endothelial layer is fenestrated (e.g., glomeruli in the kidney), or discontinuous (e.g., liver sinusoids) and less dense to allow for rapid molecular exchange. ECs not only differ between organs or vascular systems, they also change along the vascular tree and specialized subpopulations of ECs can be found within the capillaries of a single organ. Molecular tools that enable selective vascular targeting are helpful to experimentally dissect the role of distinct EC populations, to improve molecular imaging and pave the way for novel treatment options for vascular diseases. This review provides an overview of endothelial diversity and highlights the most successful methods for selective targeting of distinct EC subpopulations.
... Carcinoma-associated fibroblast CRVLRSGSC (Ruoslahti, 2014) Mouse lung CGFECVRQCPERC (Rajotte et al., 1998;Ruoslahti, 2014) Mouse skin CVALCREACGEGC (Rajotte et al., 1998) Mouse retina CSCFRDVCC (Rajotte et al., 1998) Mouse pancreas SWCEPGWCR (Rajotte et al., 1998) Mouse brain CSSRLDAC, WPFYGTP, LPSSLQK, CTSTSAPYC (Pasqualini et al., Pasqualini & Ruoslahti, 1996;Rajotte et al., 1998;Chen, Chang, & Davidson, 2009;Li et al., 2012) Mouse kidney CLPVASC (Pasqualini et al., Pasqualini & Ruoslahti, 1996;Chen et al., 2009) Bone-like mineral VTKHLNQISQSY (Pasqualini et al., Pasqualini & Ruoslahti, 1996;Rivinoja et al., Rivinoja & Laakkonen, 2011) Mouse thymus blood vessels CHAQGSAEC (Yu, Wang, & Du, 2008 (Denby et al., 2007;Wan et al., 2009) Human prostate cancer PKRGFQD, SNTRVAP (Mandelin et al., 2015) Human medullary thyroid carcinoma SRESPHP Denby et al., 2007) Human melanoma SGLYKVAYDWQH (Böckmann, Hilken, et al., 2005;Yang et al., 2018) Human ovarian cancer WSGPGVWGASVK Human gastric peritoneal tumor SWKLPPS (Akita et al., 2006) Human breast tumor AREYGTRFSLIGGYR (Qu, Qiu, Zhu, Yang, & Mao, 2017;Yang et al., 2018) Human lung adenocarcinoma CAKATCPAC (Lee et al., 2016;Qu et al., 2017) (Continues) Specifically, Mao group first identified a rat mesenchymal stem cells (rMSC)-binding peptide, VTAMEPGQ, through phage biopanning, (Ma, Wang, et al., 2013) then disassembled the VTAMEPGQ phage to collect the pVIII coat protein displaying the peptide (VTAMEPGQ-pVIII), and functionalized gene-carrying magnetic silica nanoclusters with the collected VTAMEPGQ-pVIII protein to increase the gene delivery into rMSCs. They found that the phage-borne rMSCbinding VTAMEPGQ-pVIII significantly increased the transfection efficiency up to 40% in rMSCs (Figure 3), confirming the important role of phage display technique in improving the gene delivery. ...
... Carcinoma-associated fibroblast CRVLRSGSC (Ruoslahti, 2014) Mouse lung CGFECVRQCPERC (Rajotte et al., 1998;Ruoslahti, 2014) Mouse skin CVALCREACGEGC (Rajotte et al., 1998) Mouse retina CSCFRDVCC (Rajotte et al., 1998) Mouse pancreas SWCEPGWCR (Rajotte et al., 1998) Mouse brain CSSRLDAC, WPFYGTP, LPSSLQK, CTSTSAPYC (Pasqualini et al., Pasqualini & Ruoslahti, 1996;Rajotte et al., 1998;Chen, Chang, & Davidson, 2009;Li et al., 2012) Mouse kidney CLPVASC (Pasqualini et al., Pasqualini & Ruoslahti, 1996;Chen et al., 2009) Bone-like mineral VTKHLNQISQSY (Pasqualini et al., Pasqualini & Ruoslahti, 1996;Rivinoja et al., Rivinoja & Laakkonen, 2011) Mouse thymus blood vessels CHAQGSAEC (Yu, Wang, & Du, 2008 (Denby et al., 2007;Wan et al., 2009) Human prostate cancer PKRGFQD, SNTRVAP (Mandelin et al., 2015) Human medullary thyroid carcinoma SRESPHP Denby et al., 2007) Human melanoma SGLYKVAYDWQH (Böckmann, Hilken, et al., 2005;Yang et al., 2018) Human ovarian cancer WSGPGVWGASVK Human gastric peritoneal tumor SWKLPPS (Akita et al., 2006) Human breast tumor AREYGTRFSLIGGYR (Qu, Qiu, Zhu, Yang, & Mao, 2017;Yang et al., 2018) Human lung adenocarcinoma CAKATCPAC (Lee et al., 2016;Qu et al., 2017) (Continues) Specifically, Mao group first identified a rat mesenchymal stem cells (rMSC)-binding peptide, VTAMEPGQ, through phage biopanning, (Ma, Wang, et al., 2013) then disassembled the VTAMEPGQ phage to collect the pVIII coat protein displaying the peptide (VTAMEPGQ-pVIII), and functionalized gene-carrying magnetic silica nanoclusters with the collected VTAMEPGQ-pVIII protein to increase the gene delivery into rMSCs. They found that the phage-borne rMSCbinding VTAMEPGQ-pVIII significantly increased the transfection efficiency up to 40% in rMSCs (Figure 3), confirming the important role of phage display technique in improving the gene delivery. ...
Display of a peptide or protein of interest on the filamentous phage (also known as bacteriophage), a biological nanofiber, has opened a new route for disease diagnosis and therapy as well as proteomics. Earlier phage display was widely used in protein–protein or antigen–antibody studies. In recent years, its application in nanomedicine is becoming increasingly popular and encouraging. We aim to review the current status in this research direction. For better understanding, we start with a brief introduction of basic biology and structure of the filamentous phage. We present the principle of phage display and library construction method on the basis of the filamentous phage. We summarize the use of the phage displayed peptide library for selecting peptides with high affinity against cells or tissues. We then review the recent applications of the selected cell or tissue targeting peptides in developing new targeting probes and therapeutics to advance the early diagnosis and targeted therapy of different diseases in nanomedicine. We also discuss the integration of antibody phage display and modern proteomics in discovering new biomarkers or target proteins for disease diagnosis and therapy. Finally, we propose an outlook for further advancing the potential impact of phage display on future nanomedicine.
This article is categorized under:
• Biology‐Inspired Nanomaterials > Protein and Virus‐Based Structures
... 47 While this has been attempted through IV injection, it is yet to be attempted by RU or SC injections. 48,49 AAV can also be put through ''directed evolution'' using DNA shuffling techniques to increase tropism for target tissue. 50 In a similar manner, Ad proteins can be modified to target ligands that may be present on specific cell types. ...
There are many kidney diseases that might be addressed by gene therapy. However, gene delivery to kidney cells is inefficient. This is due in part to the fact that the kidney excludes molecules above 50 kDa and that most gene delivery vectors are megaDaltons in mass. We compared the ability of adeno-associated virus (AAV), adenovirus (Ad), and lentiviral (LV) vectors to deliver genes to renal cells. When vectors were delivered by the intravenous (IV) route in mice, weak luciferase activity was observed in the kidney with substantially more in the liver. When gene delivery was observed in the kidney, expression was primarily in the glomerulus. To avoid these limitations, vectors were injected directly into the kidney by retrograde ureteral (RU), and subcapsular (SC) injections in mice. Small AAV vectors transduced the kidney, but also leaked from the organ and mediated higher levels of transduction in off-target tissues. Comparison of AAV2, 6.2, 8, and rh10 vectors by direct kidney injection demonstrated highest delivery by AAV6.2 and 8. Larger Ad and LV vectors transduced kidney cells and mediated less off-target tissue transduction. These data demonstrate the utility of direct kidney injections to circumvent the kidney size exclusion barrier. They also identify the effects of vector size on on-target and off-target transduction. This lays the foundation for the use of different vector platforms for gene therapy of diverse kidney diseases.
... Phage display in vitro and in vivo screens have identified several unique, short peptide sequences that confer tissue homing specificities [49,50]. Homing peptides targeting tissues such as lung [50,51], brain [52,53], kidney [54], muscle [55,56], and ischemic myocardium [57] have been reported. CDC-EV may potentially have clinical applications for treating muscular dystrophies in DMD [15]. ...
Background:
Extracellular vesicles (EVs) and exosomes are nano-sized, membrane-bound vesicles shed by most eukaryotic cells studied to date. EVs play key signaling roles in cellular development, cancer metastasis, immune modulation and tissue regeneration. Attempts to modify exosomes to increase their targeting efficiency to specific tissue types are still in their infancy. Here we describe an EV membrane anchoring platform termed "cloaking" to directly embed tissue-specific antibodies or homing peptides on EV membrane surfaces ex vivo for enhanced vesicle uptake in cells of interest. The cloaking system consists of three components: DMPE phospholipid membrane anchor, polyethylene glycol spacer and a conjugated streptavidin platform molecule, to which any biotinylated molecule can be coupled for EV decoration.
Results:
We demonstrate the utility of membrane surface engineering and biodistribution tracking with this technology along with targeting EVs for enhanced uptake in cardiac fibroblasts, myoblasts and ischemic myocardium using combinations of fluorescent tags, tissue-targeting antibodies and homing peptide surface cloaks. We compare cloaking to a complementary approach, surface display, in which parental cells are engineered to secrete EVs with fusion surface targeting proteins.
Conclusions:
EV targeting can be enhanced both by cloaking and by surface display; the former entails chemical modification of preformed EVs, while the latter requires genetic modification of the parent cells. Reduction to practice of the cloaking approach, using several different EV surface modifications to target distinct cells and tissues, supports the notion of cloaking as a platform technology.
... This is a molar ratio of 10 cold avidin molecules to each phage virion. In vivo phage display is a very versatile technique able to identify phage clones with various pharmacokinetic properties (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). We utilize a pre-clearing step as a negative selection to remove phage clones that bind to serum components and vascular cells. ...
Bacteriophage (phage) display technology is a powerful strategy for the identification of peptide-based tumor targeting agents for drug discovery. Phage selections performed in vitro often result in many phage clones/peptides with similar properties and often similar sequence. However, these phage and corresponding peptides are selected, validated, and characterized outside the complicated milieu of a living animal. Thus, there is no guarantee that peptides from in vitro selections will successfully meet the requirements of an in vivo targeting compound. In comparison, in vivo phage display selections have the distinct advantage of identifying phage clones with robust pharmacokinetics and tumor/tissue targeting ability. This capacity has allowed for the identification of peptides with specific in vivo localization and/or clearance profiles. However, in vivo phage display selections also have the potential to result in an array of phage clones with various and unknown targets and little to no sequence similarity. Given these shortcomings, we have developed methods to select phage peptide display libraries in living mice to identify phage (and corresponding synthesized peptides) with specific clearance and/or tumor-targeting propensity. Additionally, we describe the use of labeled phage clones for the efficient screening of selected phage/peptides to aid in the identification and characterization of a phage clone with an optimal and specific pharmacokinetic profile.
... It would appear that the constraints of the genetic approach imposing secondary structure on the targeting peptide may have reduced the affinity of the selected peptide for the target receptor, although previous strategies utilizing the same approach have yielded success. [43][44][45][46][47] A key limitation to the clinical deployment of Ad-based medicines is the high level of preexisting immunity, estimated at > 90% in some populations, which results in rapid sequestration, neutralization, and elimination of virotherapy, following systemic administration. The immunodominant epitopes remain a subject of debate, with a number of publications suggesting that Ad5 hexon hypervariable regions (HVRs) are the major site of neutralization, at least following intramuscular challenge. ...
Oncolytic virotherapies based on Adenovirus 5 (Ad5) hold promise as adjunctive cancer therapies; however their efficacy when delivered systemically is hampered by poor target cell specificity and pre-existing anti-Ad5 immunity. Ovarian cancer represents a promising target for virotherapy, since the virus can be delivered locally into the peritoneal cavity. Both Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor 1 (FGFR1) are over-expressed in the majority of human tumours, including ovarian cancer. To generate adenoviral vectors with improved tumour specificity, we generated a panel of Ad5 vectors with altered tropism for EGFR and FGFR, rather than the natural Ad5 receptor, hCAR. We have included mutations within AB loop the viral fibre knob (KO1 mutation) to preclude interaction with, hCAR, combined with insertions in the HI loop to incorporate peptides that bind either EGFR (peptide YHWYGYTPQNVI, GE11) or FGFR1 (peptides MQLPLAT, M* and LSPPRYP, LS). Viruses were produced to high titres, and the integrity of the fibre protein was validated by Western blotting. The KO1 mutation efficiently ablated hCAR interactions, and significantly increased transduction was observed in hCARlow/EGFRhigh cell lines using Ad5.GE11, whilst transduction levels using Ad5.M* or Ad5.LS were not increased. In the presence of physiological concentrations of human blood clotting factor X (hFX), significantly increased levels of transduction via the hFX-mediated pathway were observed in cell lines, but not in primary tumour cells derived from epithelial ovarian cancer (EOC) ascites samples. Ad5 mediated transduction of EOC cells were completely abolished by the presence of 2.5% serum from patients, whilst surprisingly, incorporation of the GE11 peptide resulted in significant evasion of neutralisation in the same samples. We thus speculate that incorporation of the YHWYGYTPQNVI dodecapeptide within the fibre knob domain may provide a novel means of circumventing pre-existing Ad5 immunity that warrants further investigation.
... The fi rst studies utilized Ad35 fi ber interaction with CD46 by pseudotyping onto Ad5 to bypass the challenges with transducing Ad5-refractory cells (hematopoietic cells) due to low CAR expression ( 33 ) . Furthermore, pseudotyping Ad5 with the rare species D 19p fi ber produced vectors with signi fi cantly decreased liver transduction ( 34 ) which enabled retargeting to the rat kidney following insertion of renal targeting peptides into the HI loop ( 35 ) . ...
Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by "off target" interactions that preclude cell specific targeting to sites of disease. A number of "off target" interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Factor, p-selectin as well as neutralizing antibodies. Thus, to improve efficacious targeting to sites of disease and limit nonspecific uptake of virus to non-target tissues, specifically the liver and the spleen, it is necessary to develop suitable strategies for genetically modifying the capsid proteins to preclude these interactions. To this end we have developed versatile systems based on homologous recombination for modification of each of the major capsid proteins, which are described herein.
... Several vascular-homing peptides with unknown targets have been identified in endothelium of blood-brain barrier (BBB), kidney, prostate cancer, ovarian cancer, melanoma, gastric cancer, abnormal retina neovessels, and inflamed endothelium [20,[81][82][83][84][85][86][87][88][89]. More in details, the CGNSNPKSC peptide conjugated to the recombinant mutant human TNFα (rmhTNFα) was found to selectively bind to the gastric tumor vasculature with a higher anti-tumor activity and lower systemic toxicity than the rmhTNFα alone [90]. ...
... Vector engineering has been facilitated by the resolution of the crystal structure of the Ad5 fiber knob domain (Xia et al., 1994). The fiber knob exists as a homotrimer, with the topology of an eight-stranded, anti-parallel β-sandwich, with interspersing loop regions (Xia et al., 4 4 phage display technology (Xia et al., 2000;Nicklin et al., 2001;Work et al., 2004;Denby et al., 2007). ...
... In contrast to the large number of studies which have assessed the suitability of Ad5 for selective retargeting, such approaches using rare serotype Ad vectors remain in their infancy, although fiber pseudotyping is commonly employed (Chiu et al., 2001;Nakayama et al., 2006;Parker et al., 2013). Attempts previously have been made to genetically modify the fiber of species B (Ad35) (Matsui et al., 2009), D (Ad19p fiber) (Denby et al., 2007) and F (Ad41 short fiber) serotypes (Hesse et al., 2007). The insertion of peptides within the EG, HI and IJ loops of these fiber knobs was found to be compatible with fiber trimerization and resulted in successful modification of tropism (Denby et al., 2007;Hesse et al., 2007). ...
... Attempts previously have been made to genetically modify the fiber of species B (Ad35) (Matsui et al., 2009), D (Ad19p fiber) (Denby et al., 2007) and F (Ad41 short fiber) serotypes (Hesse et al., 2007). The insertion of peptides within the EG, HI and IJ loops of these fiber knobs was found to be compatible with fiber trimerization and resulted in successful modification of tropism (Denby et al., 2007;Hesse et al., 2007). In this study, we have assessed the feasibility of peptide incorporation into the CD, DG, HI or IJ loops of the Ad48 fiber knob. ...
Ad5 is a commonly used vector for gene therapy but its efficacy is limited by high seroprevalence and off-target hepatic and splenic sequestration. In order to circumvent these limitations, the use of vectors derived from rare species adenoviruses is appealing. The opportunity to retarget rare species vectors to defined cell types through the incorporation of peptide ligands would be advantageous, particularly in targeting tumors and disseminated metastases. We used predictive structural modelling to assess the CD, DG, HI and IJ loops of the Ad48 fiber knob and identify optimal incorporation locales for the 20-mer peptide, A20FMDV2 (A20). A20FMDV2 targets ανβ6 integrin, which is overexpressed in human carcinomas. Recombinant Ad48 fiber knob proteins, Knob48, Knob48-CD-A20, Knob48-DG-A20, Knob48-HI-A20 and Knob48-IJ-A20 were engineered and purified following expression in E.coli. We confirmed that Knob48, Knob48-CD-A20 and Knob48-IJ-A20 formed stable homotrimers. However, Knob48-DG-A20 and Knob-HI-A20 failed to form a trimer. All A20-modified knob proteins blocked the transduction of Ad5-EGFPA20 via ανβ6, demonstrating that the inserted A20 peptide was functional. In conclusion, we show that the CD and IJ loops of Ad48 represent suitable sites for targeting peptide incorporation. Interestingly, in vitro gene transfer mediated by the non-FX binding Ad48 vector was not sensitive to immunoglobulins and complement when incubated in the presence of mouse serum, unlike Ad5. These data support the future generation of the corresponding Ad48 viral vectors, Ad48-CD-A20 and Ad48-IJ-A20, which may offer favourable characteristics for targeted delivery in vivo.
