Intramuscular Injection Of AAV8 In Mice And Macaques Is .
Intramuscular Injection of AAV8 in Mice and Macaques IsAssociated with Substantial Hepatic Targeting andTransgene ExpressionJenny A. Greig, Hui Peng, Jason Ohlstein, C. Angelica Medina-Jaszek, Omua Ahonkhai, Anne Mentzinger,Rebecca L. Grant, Soumitra Roy, Shu-Jen Chen, Peter Bell, Anna P. Tretiakova, James M. Wilson*Gene Therapy Program, Department of Pathology and Laboratory Medicine, Division of Transfusion Medicine, University of Pennsylvania, TRL Suite 2000, 125 South 31stStreet, Philadelphia, PA, 19104, United States of AmericaAbstractIntramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinicaldevelopment with some success, including the first approved gene therapy product in the West called Glybera. Inpreparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies inmice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administrationof AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly tototal expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and bythe use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements.Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAVdelivery should inform safe and efficient development of future AAV products.Citation: Greig JA, Peng H, Ohlstein J, Medina-Jaszek CA, Ahonkhai O, et al. (2014) Intramuscular Injection of AAV8 in Mice and Macaques Is Associated withSubstantial Hepatic Targeting and Transgene Expression. PLoS ONE 9(11): e112268. doi:10.1371/journal.pone.0112268Editor: Knut Stieger, Justus-Liebig-University Giessen, GermanyReceived March 31, 2014; Accepted October 6, 2014; Published November 13, 2014Copyright: ß 2014 Greig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.Funding: This work was supported by the Bill & Melinda Gates Foundation, grant number 51061 and URL http://www.gatesfoundation.org/. The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: J.M. Wilson is an advisor to ReGenX Biosciences and Dimension Therapeutics, and is a founder of, holds equity in, and receives grantsfrom ReGenX Biosciences and Dimension Therapeutics; in addition, he is a consultant to several biopharmaceutical companies and is an inventor on patentslicensed to various biopharmaceutical companies. Numerous patents describing the isolation of various AAVs and their use in gene therapy have originated fromthe Gene Therapy Program. Several patents and applications describe methods of producing AAV vectors. Some patents have issued, and some are underprosecution. Our portfolio relating to AAV vectors spans over 200 patents and applications worldwide. This does not alter the authors’ adherence to PLOS ONEpolicies on sharing data and materials. All other authors declare no competing interests.* Email: wilsonjm@mail.med.upenn.eduhundred 1.35 ml vector injections IM spread across ten sites or upto sixty 0.5 ml injections in clinical trials for Glybera [6,11].Local injection of vector into most tissues could lead to apercentage of the injected volume disseminating from the site ofinjection and being transported to other organs. We speculate thatthe larger the volume of an IM injection, potentially the greaterfraction of the volume can be dispersed from the site ofadministration. Therefore, due to the natural or increased tropismof certain AAV vectors for the liver and the resulting livertransduction, a significant contribution to the total level of asecreted transgene protein may be contributed by the liverfollowing IM administration. Also, distribution of vector beyondthe muscle could have implications on the safety and immunogenicity of the treatment. It has been suggested that delivery of thevector to liver, either by design or inadvertently, could induceimmunologic tolerance to the transgene product, thereby diminishing immune toxicity [16,17,18,19,20,21,22,23,24]. In contrast,IM administration of concentrated AAV vectors, resulting in highvector dose per injection site, has been linked to higher levelantibody production against the secreted transgene product [4,5].An AAV product can be engineered to restrict the expression oftransgenes following different systemic routes of administration,IntroductionVectors derived from adeno-associated viruses (AAV) have beenshown to produce long-term and stable gene expression of secretedproteins in a variety of animal models and human clinical trialsfollowing intramuscular (IM) injection, including coagulationfactor IX (FIX) [1,2,3,4,5], alpha-1-antitrypsin (AAT) [6,7],erythropoietin [8], and neutralizing immunoglobulins againstHIV [9,10]. Intramuscular (IM) delivery of an AAV vectorprovides a quick, easy, non-invasive and safe route of administration, which can be routinely performed in virtually any setting.The most celebrated example of IM AAV gene therapy is thetreatment of an inherited deficiency of lipoprotein lipase with thecommercially approved product Glybera [11]. However, previousstudies have identified some of the limitations of IM injections,whereby transduction is limited to cells around the needle tractarea of the injection site in mice, nonhuman primates (NHP) andhumans [3,12,13,14,15]. This has led to the practice of a largenumber of small volume IM injections to produce sufficienttransgene expression [7]. For example, subjects enrolled in thehigh dose cohorts of the Phase II AAT clinical trial received onePLOS ONE www.plosone.org1November 2014 Volume 9 Issue 11 e112268
AAV8 Hepatic Targeting following IM Injectionsuch as IM, which will lead to broad distribution of vector. Themore traditional approach to overcome this problem is to driveexpression of the transgene from a tissue-specific promoter, such asthe muscle creatine kinase (tMCK) promoter for skeletal muscleexpression [25] and the human thyroxine binding globulin (TBG)promoter for liver expression [26,27]. Transgene expression canalso be inhibited in certain organs by the incorporation of tissuespecific microRNA target sites [28,29]. Interaction of microRNAswith their complementary target sites within the RNA-inducedsilencing complex can lead to inhibition of translation ordegradation of mRNA [30,31]. Incorporation of 3–6 copies oftarget sites for the liver-specific microRNA (miR) 122 or skeletalmuscle-specific miR-206 in the 39 UTR of an AAV vector hasbeen previously shown to reduce liver and muscle gene expression,respectively [32,33,34,35]. Therefore, transgene expression couldbe restricted from either liver by miR-122 or muscle by miR-206.In the current study we have evaluated important aspects ofAAV8 IM delivery, such as concentration and volume, andfeatures of the expression cassette, such as tissue specificity, onsafety and efficacy in mice and macaques.ResultsSubstantial gene expression from liver following IMadministration of AAV8 vectors in miceAAV8 vector expressing firefly luciferase (ffLuc) from theubiquitous CMV promoter was injected IM into C57BL/6 mice ata dose of 1010 genome copies (GC) per mouse (Figure 1A). Vectorwas administered as one 10 ml injection into the right gastrocnemius muscle and on day 7 post-vector mice were imaged todetermine the localization of ffLuc expression. Significant ffLucexpression, which localized to both the injected muscle and theliver, was demonstrated (Figure 1A). These imaging results werequantitatively reproduced and further expanded using biochemicalassays where ffLuc was measured in both liver and muscle samplesand normalized to total protein on day 28 post-vector administration (Figure 1C). IM injection of vector using the CMVpromoter for expression resulted in extensive ffLuc expression inthe injected muscle with concordant expression in the liver, whichwas 321-fold over background (control un-injected mice).The unexpectedly high levels of liver transduction after IMinjection suggested that IM delivery could be an alternative to thestandard way of targeting liver, which is by intravenous (IV)injection. Experiments were repeated with a vector expressingffLuc from the highly potent, liver-specific promoter TBG [26,27].Mice were injected IM with 1010 GC of AAV8.TBG.ffLuc in avolume of 10 ml and at day 7 post-vector administrationsignificantly higher liver expression was seen with little to no geneexpression in muscle (Figure 1B). ffLuc tissue assays on liver andmuscle taken at day 28 following administration of vector showedan increase in liver ffLuc expression of 69-fold, relative to thatachieved with the CMV promoter (Figure 1C). While muscleffLuc expression was over background (control un-injected mice)following IM injection, the three log reduction in muscleexpression seen was expected due to the specificity of the TBGpromoter (Figure 1C).As ffLuc expression allowed tissue localization of geneexpression, the net impact of inadvertent liver delivery of AAVfollowing IM injection was studied by expression of a secretedprotein. As antibodies expressed from AAV are being developed toprevent infections, including HIV and influenza [9,10,36,37],these initial studies used a gene encoding 201Ig IA. Thisimmunoadhesin (IA) construct was based on the 201Ig FAb,which was isolated from a long-term non-progressing rhesusPLOS ONE www.plosone.orgFigure 1. Liver expression following IM vector administrationin mice. Visualization of differential ffLuc expression patterns usingXenogen whole-body bioluminescent imaging on day 7 post-IMadministration of 1010 GC AAV8.CMV.ffLuc (A) or AAV8.TBG.ffLuc (B)to C57BL/6 mice. Vector was administered as one 10 ml injection intothe gastrocnemius muscle of the right leg. (C) ffLuc expression wasquantified at day 28 post-vector administration by ffLuc tissue assays.ffLuc expression measured as RLU normalized to the total proteinconcentration of the organ. Data are presented as fold change overbackground where background was RLU/total protein of organ (g) incontrol tissues from un-injected mice. (D) Comparison of expression of201Ig IA from the CMV and TBG promoters following a 10 ml IMinjection in RAG KO mice. Expression of 201Ig IA in serum wasmeasured by ELISA. Values are expressed as mean 6 SEM (n 68.g001macaque six years post-challenge with SIVsmF236 [38,39]. RAGKO mice were injected IM with 1010 GC of AAV8 expressing201Ig IA from the CMV or TBG promoters (Figure 1D).2November 2014 Volume 9 Issue 11 e112268
AAV8 Hepatic Targeting following IM InjectionA dose-dependent increase in transgene expression was seen forall AAV8 vectors, independent of route of administration orpromoter, where expression increased on average 8.5-fold acrossall transgenes when vector dose per mouse was increased by onelog (Figure 2). Substantial differences were achieved whencomparing expression from the CMV versus TBG promoter ofvectors administered IM. A statistically significant increase inexpression was observed with the TBG promoters, compared tothe CMV promoters, at the highest dose of vector with the threemAb cassettes (Figures 2B-D). A direct comparison of IM versusIV injection of the TBG promoted vectors revealed little differencein expression of the antibodies at either dose of vector (Figures 2BD). The exception to this was 201Ig IA, which was significantlyincreased (4.8-fold) following IV injection (Figure 2A). Therefore,a comparable level of secreted gene expression from a liver-specificpromoter can be produced following a quick, easy and noninvasive injection into skeletal muscle or an invasive IV injection,which requires a higher level of skill.Immunodeficient RAG KO mice were used to evaluate expressionof the 201Ig IA transgene in the absence of an immune response tothe secreted protein. Substantial levels of IA expression weredetected in blood with both vectors, although the liver-specificpromoter yielded an almost 8-fold higher expression of thesecreted protein in blood. This suggests that a substantial amountof the secreted IA is derived from liver, rather than muscle, afterIM injection. Additional studies to evaluate the relative contribution of the two tissues/organs to blood levels of the transgeneproduct are described below.Dose and route of administration impacts on expressionin miceAAV8 vectors expressing anti-SIV/SHIV antibodies (in animmunoadhesin format [i.e., 201Ig IA] or a monoclonal antibodyformat [i.e., 2.10A mAb] [40]) or human anti-HIV antibodies in amonoclonal antibody format (i.e., VRC01 mAb [41,42] and PG9mAb [43]) were injected IM as two 15 ml injections at doses of 1010GC or 1011 GC into RAG KO mice (Figure 2). Expression ofthese proteins in serum was determined on day 28 post-vectoradministration and compared to expression from TBG-containingvectors administered intravenously (IV) via the tail vein. Figure 2summarizes data from these experiments, in which the two dosesof vectors (labeled 0.1 and 1) were evaluated in the context of twocomparisons: the CMV versus TBG vectors following IM injectionand the TBG vector following IM and IV injection.Modulation of liver and muscle gene expression by IMinjection volume in miceWe investigated the impact of injection volume on the level anddistribution of transgene expression. In these studies the same doseof vector was injected in a range of volumes from two 25 mlinjections, one into each leg for a total injection volume of 50 ml, toone 2 ml injection in one site representing a 25-fold range of vectorconcentrations. The initial studies focused on mice injected withFigure 2. Comparison of expression from the liver-specific TBG promoter following IM and IV vector administration in RAG KOmice. Expression on day 28 post-vector administration of 201Ig IA (A), 2.10A mAb (B), VRC01 mAb (C), and PG9 mAb (D) in RAG KO mice. Expressionfollowing IM injection of vectors with either the CMV or TBG promoter was compared to expression levels produced following IV injection of the TBGvector. Mice were injected IM with two 15 ml injections into the right and left gastrocnemius muscles. IV injections were performed as a 100 mlinjection via the tail vein. Mice were administered with a dose of either 1010 GC or 1011 GC, numbers indicate dose 61011 GC. ND, not determined.Expression was measured in serum by ELISA and values are expressed as mean 6 SEM (n 3/group). OS ONE www.plosone.org3November 2014 Volume 9 Issue 11 e112268
AAV8 Hepatic Targeting following IM Injection1010 GC AAV8 vectors expressing ffLuc from the CMV or TBGpromoters. Tissues were harvested at day 28 and transgeneexpression measured in lysates from liver (Figure 3A) and muscle(Figure 3B). Our original hypothesis was that increasing thevolume for a fixed dose would increase the relative distribution ofvector to liver. These studies did confirm the pilot experimentsdescribed in Figure 1, which used a single concentration of vector,whereby: 1) IM injection of vector resulted in substantial levels oftransgene expression in liver, and 2) vectors using the TBGpromoter produced levels of transgene product in liver followingIM injection that were almost equivalent to the levels achievedwhen the same vector was injected IV. However, we weresurprised to see that the more concentrated IM injections of vectordid not help restrict expression to muscle; in fact these studiesshowed higher levels of liver ffLuc expression with lower volumesof vector. Also, the higher concentrations of vector yielded higherlevels of overall ffLuc in both muscle and liver, independent ofpromoter. Based on in vitro experiments (data not shown), therewas no significant loss of vector following dilution to differentconcentrations prior to administration in mice. Therefore, all micereceived the same dose of vector and differences in distribution ofthe vector were due to the concentration of the injected vector.The impact of IM injection volume was also studied in miceinjected with AAV8 vectors expressing 201Ig IA from either theCMV (Figure 3C) or TBG promoter (Figure 3D) with the readoutbeing serum levels of 201Ig IA. The CMV vectors (Figure 3C)showed dramatically lower overall expression as compared to theTBG vectors (Figure 3D). Serum 201Ig IA levels were notmarkedly affected by the volume of vector injected IM. Althoughthe highest expression from the IM injected TBG vector wasachieved with the lowest volume, it was still around two-fold lowerthan that achieved following IV injection.A series of studies were performed using LacZ as a reporter geneto evaluate distribution of transduction at a cellular level as afunction of vector concentration and dose. C57BL/6 mice wereinjected IM with AAV8 expressing LacZ from the CMVpromoter. Liver and muscle tissue were harvested for analysis byLacZ histochemical staining 21 days post-vector administration(Figure 4). Sections of the gastrocnemius muscles were taken atintervals throughout the entire injected muscle. At a dose of 1010GC, IM injection of vector as either two 25 ml injections(Figure 4A) or one 2 ml injection (Figure 4C) produced similarpatterns of expression throughout the injected muscle, with a fewtransduced cells being seen in the liver (Figures 4B, 4D); note thatthe CMV promoter does not express well in liver. To determine ifgene expression was saturated in the muscle at a dose of 1010 GCper mouse, the dose was lowered to 109 GC per mouse and a verydifferent transduction pattern was seen for the two injectionvolumes (Figures 4E, 4G). Injection of the vector IM as two 25 mlinjections produced few transduced cells in the muscle (Figure 4E).Figure 3. Reduction in IM injection volume increases transgene expression. 1010 GC AAV8 expressing ffLuc from the CMV or TBG promoterwas delivered IM as either two 25 ml injections (one into each leg), one 10 ml injection or one 2 ml injection to C57BL/6 mice. IV injections wereperformed as a 100 ml injection via the tail vein. ffLuc tissue assays were performed on tissue harvested at day 28 and normalized to the total proteinconcentration of liver (A) and muscle (B). RAG KO mice were administered with 1010 GC AAV8.CMV.201Ig IA (C) or AAV8.TBG.201Ig IA (D) by IV or IMinjections, performed as described previously. Data for the one 10 ml injection groups were previously presented in Figure 1. Expression of 201Ig IA inserum was measured by ELISA. Values are expressed as mean 6 SEM (n 4/group). *p,0.05, **p,0.01, OS ONE www.plosone.org4November 2014 Volume 9 Issue 11 e112268
AAV8 Hepatic Targeting following IM InjectionWhen the concentration of the vector was increased to enableinjection of the same dose of vector in a volume of 2 ml, a largearea of transduced skeletal muscle cells were seen (Figure 4G).This transduced area extended through multiple muscle sections.Again, only a few transduced cells were seen in the liver at a vectordose of 109 GC per mouse (Figures 4F, 4H). Therefore,administration of the same vector dose as a smaller injectionvolume improved skeletal muscle transduction at lower overallvector doses.knockdown of gene expression in the liver and muscle of 6.6and 112-fold, respectively (Figures 5A-D). As previously demonstrated [32,33,35,44,45,46], expression of the transgene in theorgan that was
studies have identified some of the limitations of IM injections, whereby transduction is limited to cells around the needle tract area of the injection site in mice, nonhuman primates (NHP) and humans [3,12,13,14,15]. This has led to the practice of a large number of small volume IM injections
Self-Injection . What is an Intramuscular Injection? An intramuscular injection, as illustrated in the figure below, delivers medication deep into the muscle tissue. This allows the medication to be quickly absorbed into the bloodstream f
injection) Code injection attacks: also known as "code poisoning attacks" examples: Cookie poisoning attacks HTML injection attacks File injection attacks Server pages injection attacks (e.g. ASP, PHP) Script injection (e.g. cross-site scripting) attacks Shell injection attacks SQL injection attacks XML poisoning attacks
Administer the injection according to the local guidelines for performing large volume intramuscular injections. NOTE: Due to the proximity of the underlying sciatic nerve, caution should be taken if administering FASLODEX at the dorsogluteal injection site [see . Warnings and Precautions (5.3) and . Adverse Reactions (6.1)].File Size: 2MB
SOP: INTRAMUSCULAR INJECTIONS IN SWINE 2 Needle Selection Guide for Intramuscular Injections (adapted from National Hog Farmer) Size of Pig Gauge Length Baby Pigs (up to 25kg) 18 or 20 5/8 to ½ inch Grower (25 to 70kg) 16 or 1
Jun 26, 2021 · Resume economic, educational, and other societal-level endeavors. Understanding Herd Immunity Vaccination or infection provide immunity . MECHANISM mRNA mRNA Adenovirus vector ADMINISTRATION Intramuscular Intramuscular Intramuscular STORAGE
cyanocobalamin injection usp. baclofen injection lidocaine hci injection (1%, 2%) piperacillin and tazobactam for injection dimenhydrinate injection usp with preservative. dimenhydrinate injection usp diazepam injection usp. naloxone hydrochloride
Diesel fuel-injection systems: An overview Fields of application 4 Technical requirements 4 Injection-pump designs 6 Mechanically-controlled (governed) axial-piston distributor fuel-injection pumps VE Fuel-injection systems 8 Fuel-injection techniques 9 Fuel supply and delivery 12 Mechanical engine-speed control (governing) 22 Injection timing 29
6 NIGHT TIME ECONOMY BLUEPRINT GREATER MANCHESTER WILL BE ONE OF THE BEST PLACES IN THE WORLD TO GO OUT, STAY OUT, WORK AND RUN A BUSINESS BETWEEN THE HOURS OF 6PM AND 6AM. We will celebrate the unique offer of each of our ten districts, recognising the importance of the night time economy to the vibrancy of our towns, cities and high streets. Our restaurants, bars, clubs and cultural .
