Podcasts about raav

Ivar on korporatiivmaailmas karjääri teinud juht, kes hoolimata kõigist täide viidud unistustest avastas ühel hetkel, et ihaldatud õnn ei seisnegi heas palgas ja mõjukas positsioonis. Läbides sügava enesesse vaatamise perioodi, ühendab ta koolitajana inimeseks olemise kaudu juhtide ja alluvate maailma. Tema meeskonnas töötanud 60 juhti ja 2000 spetsialisti on näinud tema kasvamist juhina, edu ja läbikukkumisi. Ivar on õppinud matemaatikaõpetajaks, organisatsioonipsühholoogiat ja teraapiat, ehitanud üles mitmetest lähenemistest oma viisi, kuidas töökohtades inimesed rohkem iseendid saaksid olla, ning toob selles raamatus välja oma olulisimad põhimõtted, et juhtimiskvaliteet tõuseks tasemele, kus me teame, mida oodata juhtidelt, ja julgeksime seda ootust ka väljendada. Mitte keegi ei tohi aktsepteerida kehva juhtimist, on Ivari üks juhtivaid põhimõtteid. Kõige enam rõhku soovib Ivar suunata just sellele, et kuidas korporatiivmaailmas tegutsemine käivitas temas sügavama vajaduse ja tahte iseendasse vaadata, kuna pealtnäha oli kõik suurepärane, aga tegelikult sisimas toimus täielik kaos. Kuidas teiste aitamise soovi tulemusena sai iseennast ära lõhutud. Ivar on andnud välja ka raamatu nimega “Juhtimise jalajälg - miks peaks juhtimine olema ajutine”, kus ta räägib lahti oma põhimõtted ja kogemuse, kuidas ühendada maailmad, kus ei ole traditsioonilist juhtimist, vaid on suhe ja side inimeste vahel. Lisaks veab ta ka podcasti “Juhtimine juhtimiseta”. Saates räägime Ivariga sellistel teemadel nagu juht versus palgatöötaja, töökultuur ja organisatsioonipsühholoogia, ühiste eesmärkide suunas liikumine tiimina, Ivari enda isiklik areng ja välja astumine juhtimisest, kellest saab hea juht, kes on määratud olema kehv juht, holistilisest regressiooniteraapiast, mentordamisest jne. Saatest saad teada: - kuidas ja miks on juhid kõige suuremad organisatsioonide kultuuride mõjutajad; - Ivari enda teekond läbi enesearengu; - kas sinust võiks saada hea juht; - kuidas jõuda sügavama kontakti iseendaga; - kuidas iseendaga sügavam kontakt aitab luua paremat jalajälge maailmas; https://ivarraav.com/

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.536004v1?rss=1 Authors: Root, J., Mendsaikhan, A., Nandy, S., Taylor, G., Wang, M., Troiano Araujo, L., Merino, P., Ryu, D., Holler, C., Thompson, B. M., Astarita, G., Blain, J.-F., Kukar, T. Abstract: Progranulin (PGRN) deficiency is linked to neurodegenerative diseases including frontotemporal dementia, Alzheimers disease, Parkinsons disease, and neuronal ceroid lipofuscinosis. Proper PGRN levels are critical to maintain brain health and neuronal survival, however the function of PGRN is not well understood. PGRN is composed of 7.5 tandem repeat domains, called granulins, and is proteolytically processed into individual granulins inside the lysosome. The neuroprotective effects of full-length PGRN are well-documented, but the role of granulins is still unclear. Here we report, for the first time, that expression of single granulins is sufficient to rescue the full spectrum of disease pathology in mice with complete PGRN deficiency (Grn-/-). Specifically, rAAV delivery of either human granulin-2 or granulin-4 to Grn-/- mouse brain ameliorates lysosome dysfunction, lipid dysregulation, microgliosis, and lipofuscinosis similar to full-length PGRN. These findings support the idea that individual granulins are the functional units of PGRN, likely mediate neuroprotection within the lysosome, and highlight their importance for developing therapeutics to treat FTD-GRN and other neurodegenerative diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Synopsis: Joel Schneider, Ph.D. and Robert Kotin, Ph.D. are the President & CEO and Founder & Chief Technology Advisor, respectively, of Carbon Biosciences, an emerging leader in the development of novel parvovirus-derived gene therapies. Joel and Robert sit down with host Rahul Chaturvedi to discuss the arc of their careers, how the AAV field has changed over the last decade, and how they approach building out their team at Carbon. They also talk about Carbon's focus on cystic fibrosis and the implications of The Cystic Fibrosis Foundation investing in their Series A. Finally, they both weigh in on what excites them when thinking about the future of gene therapy. Biographies: Dr. Joel Schneider joined Carbon as CEO in 2022, after serving as Chief Operating Officer at Solid Biosciences. As Solid's first employee, he played an instrumental role in building the company's unique disease-focused business model. Dr. Schneider is an accomplished biosciences executive with a track record of achievement in identifying, developing, and financing high potential therapeutic modalities and has diverse leadership experiences across R&D, technical and corporate operations, and corporate development organizations. Dr. Schneider holds a Ph.D. from Rutgers University and an undergraduate degree from Brandeis University and is the author of numerous peer-reviewed articles related to Duchenne and stem cell biology. He completed a postdoctoral fellowship at Harvard University in the Department of Stem Cell and Regenerative Biology, where he characterized and developed the small molecules that enhance skeletal muscle regeneration. Dr. Robert Kotin has been a leader in adeno-associated virus (AAV) research for 35 years, focusing on the molecular biology of the virus's non-structural proteins and then leveraging this understanding to develop novel AAV vectors for somatic cell gene therapy. Beginning as a postdoctoral fellow at Cornell University Medical Center, Dr. Kotin discovered a common integration site for AAV DNA in human chromosome 19, which he designated AAVS1 locus. He spent most of his career in the Intramural Research Program at the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH), first as a tenure-track investigator and then as a tenured senior investigator and Head of the Laboratory of Molecular Biology and Gene Therapy. While at the NIH, Dr. Kotin's laboratory invented and developed a scalable recombinant adeno-associated virus (rAAV) production process in Sf9 cells which was licensed by UniQure, ThermoFisher, Voyager, Biomarin, and others and was used to produce Glybera™, the first rAAV product granted regulatory agency approval for sale. Additional research from the Kotin lab resulted in the discovery of an AAV replicative product that has been described as closed-ended linear duplex DNA (ceDNA) and became the basis of the non-viral gene therapy company Generation Bio (NASDAQ: GBIO). Dr. Kotin served as vice president of virology and gene therapy at Voyager Therapeutics from 2014 to 2016. Since 2016, he has served as an adjunct professor at UMass Medical School, where his research interests include vectorizing and characterizing ancestral parvoviruses based on inferred sequences from endogenous virus elements (EVEs) as novel gene therapy vectors. Dr. Kotin earned his B.A. in biology from the University of California, Santa Cruz, and his doctorate in microbiology from Rutgers University and the University of Medicine and Dentistry of New Jersey (now Robert Wood Johnson Medical School).

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.08.519609v1?rss=1 Authors: Hovde, K., Rautio, I. V., Hegstad, A. M., Witter, M. P., Whitlock, J. R. Abstract: The mammalian visual system can be broadly divided into two functional processing pathways: a dorsal stream supporting visually and spatially guided actions, and a ventral stream enabling object recognition. In rodents, the majority of visual signaling in the dorsal stream is transmitted to frontal motor cortices via extrastriate visual areas surrounding V1, but exactly where and to what extent V1 feeds into motor-projecting visual regions is not well known. To address this we employed a dual labeling strategy in male and female mice in which efferent projections from V1 were labeled anterogradely, and motor-projecting neurons in higher visual areas were labeled with retrogradely traveling adeno-associated virus (rAAV-retro) injected in M2. In flattened sections of dorsal cortex, the most pronounced colocalization V1 output and M2 input occurred in extrastriate areas AM, PM, RL and AL. Coronal sections further showed that neurons in both superficial and deep layers in these regions project to M2, but high resolution volumetric reconstructions revealed that the vast majority of putative synaptic contacts from V1 onto M2-projecting neurons occurred in layer 2/3. These findings support the existence of a dorsal processing stream in the mouse visual system, where visual signals reach motor cortex largely via feedforward projections in anteriorly and medially located extrastriate areas. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Wieso die Kombination aus Yoga, Akupressur, Faszialer und Viszeraler Behandlung, Energetik und Ernährung den optimalen Weg für tiefe Heilung ebnet. Wir sprechen mit Tim Raav über seine neu entwickelten Therapieformen wie Biofunktionaltherapie. In Erweiterung sein YouTube Kanal, seine App, seine eigene Akademie und weitere Produkte für zuhause wie er zahlreiche Menschen damit erreicht. YouTube: https://m.youtube.com/watch?v=B2USQmNPpyE Acuflow - Tools for Treatment: https://acuflow.de App IOS https://apps.apple.com/at/app/raav-therapie/id1479682233 Herzlich Willkommen beim Gesundheitspodcast vom ALPHAtauern Health Resort. Monatlich treffen wir unterschiedlichste Expert*innen und stellen Fragen zu Themen aus Wissenschaft, Sport und Medizin. Wir suchen Antworten auf die Fragen „Was macht ein gesundes Leben aus?“ oder „Was kann ich dafür tun, möglichst lange fit zu bleiben?“ und vielleicht lässt sich mit diesem Wissen am Ende ja sogar ein längeres Leben führen. Bei Fragen, Wünsche oder Anregungen rund um unseren Podcast stehen wir Euch gerne unter der E-Mail Adresse: bernhard@alphatauern.at zur Verfügung. Hinterlasst uns doch gerne eine Bewertung und Rezension auf den Plattformen. Herzlichen Dank & stay healthy

Transient transfection of plasmids into suspension HEK293 cells is still the most commonly used method for the production of recombinant adeno-associated virus (rAAV) gene therapies – in this episode, Diane Golebiowski and Marissa Stanvick discuss ways to optimize that process. 

In this episode of Talking Techniques, supported by Bio-Rad, we discuss a key component of many gene therapies: recombinant adeno-associated viruses (rAAVs) and their production. These viruses act as efficient, accurate delivery vesicles for the gene therapy's plasmid. Speaking to Associate Director of Biopharma Product Marketing at Bio-Rad Laboratories, Mark White, we take a look the different expression systems used for their production and compare their advantages, before looking at some of the challenges involved in the production rAAVs, such as host cell contamination. Discover the tools that can help minimize host cell contamination and differentiate between nuclease resistant and nuclease reactive contaminant DNA and find out about some of the most exciting developments in rAAV technologies.   Contents:The role of rAAVs in gene therapies: 00:40-02:15 The production of rAAVs and gene therapies: 02:15-03:30 Why are HEK cells so popular for cell therapy production? 03:30-05:45 HEK vs SF9 Insect cell expression systems: 05:45-06:45 Challenges in cell therapy expression systems: 06:45-08:05 Host DNA contamination: 08:05-10:30 The risks of host DNA contamination: 10:30-12:45 Key techniques to minimize host DNA contamination: 12:45-14:40 The advantages of ddPCR in gene therapy production: 14:40-17:50 Distinguishing between nuclease resistant and nuclease reactive host cell DNA: 17:50-19:10 The most exciting developments in rAAV technology: 19:10-20:20 What is one thing you would ask for to improve rAAV and gene therapy development 20:20-26:38

In this episode of Talking Techniques, supported by Bio-Rad, we discuss a key component of many gene therapies: recombinant adeno-associated viruses (rAAVs) and their production. These viruses act as efficient, accurate delivery vesicles for the gene therapy's plasmid.Speaking to Associate Director of Biopharma Product Marketing at Bio-Rad Laboratories, Mark White, we take a look at the different expression systems used for their production and compare their advantages, before looking at some of the challenges involved in the production rAAVs, such as host-cell contamination.Discover the tools that can help minimize host-cell contamination and differentiate between nuclease resistant and nuclease reactive contaminant DNA and find out about some of the most exciting developments in rAAV technologies. Contents:The role of rAAVs in gene therapies: 00:40-02:15The production of rAAVs and gene therapies: 02:15-03:30Why are HEK cells so popular for cell therapy production? 03:30-05:45HEK vs SF9 Insect cell expression systems: 05:45-06:45Challenges in cell therapy expression systems: 06:45-08:05Host DNA contamination: 08:05-10:30The risks of host DNA contamination: 10:30-12:45Key techniques to minimize host DNA contamination: 12:45-14:40The advantages of ddPCR in gene therapy production: 14:40-17:50Distinguishing between nuclease resistant and nuclease reactive host cell DNA: 17:50-19:10The most exciting developments in rAAV technology: 19:10-20:20What is one thing you would ask for to improve rAAV and gene therapy development 20:20-26:38 See acast.com/privacy for privacy and opt-out information.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.08.139667v1?rss=1 Authors: gong, h., Yuan, N., Shen, Z., Tang, C., Shipp, S., Qian, L., Lu, Y., Andolina, I. M., Zhang, S., Wu, J., Yang, H., Wang, W. Abstract: Rapid and efficient gene transduction via recombinant adeno-associated viruses (rAAVs) is highly desirable across many basic and clinical research domains. Here we report vector co-infusion with doxorubicin, a clinical anti-cancer drug, markedly enhanced rAAV-mediated gene expression in cerebral cortex across mammalian species (cat, mouse, and macaque), acting throughout the time-period examined and detectable at just three days post-transfection. This enhancement showed serotype generality, being common to rAAV serotypes 2, 8, 9 and PHP.eB tested, and was observed both locally, and at remote locations consistent with doxorubicin undergoing retrograde axonal transport. All these effects were observed at doses matching human blood plasma levels in clinical therapy, and lacked detectable cytotoxicity as assessed by cell morphology, activity, apoptosis and behavioral testing. Altogether, this study identifies an effective means to improve the capability and scope of in vivo rAAV applications, accelerating and augmenting gene transduction at doxorubicin concentrations paralleling medical practice. Copy rights belong to original authors. Visit the link for more info

Minu seekordseks vestluskaaslaseks on juhtimispraktik ja –koolitaja Ivar Raav. Ta on juhina töötanud kümmekond aastat nii Swedbankis, Omnivas kui ka If Kindlustuses. Mind ajendas Ivariga rääkima see, et ta on toonud meie juhtimisleksikoni sellise mõiste nagu ‘juhtimise jalajälg’. Juhtimisjälg viitab muutustele, mille juht jätab endaga koostööst meeskonnakaaslastesse ja organisatsiooni tervikuna. Ivariga räägimegi, kuidas juhina teadlikumalt toimetada, et kujundada seda pärandit või nö ‘jälge’, mille juhina jätad endaga koostööst tiimikaaslastesse. “Juhtimise jalajälje metafoori mõte sai alguse minu jaoks ökoloogilise jalajälje mõistest ja kogu kliimatemaatikast ning ma hakkasin neid kahte asja seostama. Nii nagu ökoloogilise jalajälje puhul peaksime endalt küsima, et kuidas jätta endast maha võimalikult väike jalajälg, siis mulle tundus, et juhtidena peaksime üha rohkem endalt hakkama küsima, mis on see minu positiivne juhtimisjalajälg, mille ma tahan endast meeskonda maha jätta? Kõigepealt tuleb endale aru anda, et sa ei ole selle tiimi juht elu lõpuni. Kuigi meil võib olla tähtajatu leping juhi rollis olles, siis elu üldiselt on ikkagi selline, kus sa tead, et varem või hiljem sinu roll muutub. Juhi rolli lõplikkuse teadvustamine esitab ka küsimuse, mida ma siis päriselt soovin sellesse tiimi endast pärandina maha jätta? Mida sa soovid selle aja jooksul korda saata? Milliseks peaks su tiim kasvama või arenema, et sa saaksid südamerahus edasi liikuda? See on hea koht, kust juhina oma soovitud jalajälje teemat lahti hakata arutama. Üks pool jalajäljest on kindlasti saavutused ja äritulemused, kuid tulemustest veelgi olulisem vast on see, millise juhtimiskäekirjaga need tulemused saavutati ning millisteks inimesteks meeskonnaliikmed seeläbi kasvasid.” – Ivar Raav Kuulake ikka ...

Eduard Ayuso, INSERM 1089, University of Nantes, Nantes, France speaks on "Unraveling mechanisms and biology of recombinant AAV vectors produced in insect cells". Recombinant adeno-associated vectors (rAAV) are viral vectors of choice for gene therapy of many inherited diseases. Medicinal products based on rAAV are predominantly manufactured by transient transfection of mammalian cells or baculovirus expressing vectors (BEV) infection of insect cells, being the latter method more suitable for large-scale production. Although AAVs are mammalian viruses they can be assembled in insect cells, but the biology of the system has been poorly investigated. Here, we have studied the role of the assembly-activating protein (AAP) in insect cells and we found that this protein is expressed in a similar manner as in mammalian cells. By knocking down the AAP, it was confirmed that AAP is required for the assembly of AAV2 particles in insect cells. Next, we aimed to identify and characterize DNA species encapsidated in rAAV stocks produced in insect cells. To this end, we developed a single-strand virus sequencing protocol based on Illumina high-throughput sequencing technology (HTS). Preliminary data obtained from rAAV stocks puri ed by CsCl ultracentrifugation or immunoaffnity chromatography revealed that baculoviral and cellular DNA correspond to ≤1.5% and ≤0.02% of the total reads, respectively. Moreover, the sequencing coverage showed that the proximity to the ITRs increases progressively the probability for baculoviral DNA to be encapsidated. Nonetheless, these baculovirus-derived reads are found at a frequency of 2-3 logs lower than the rAAV genome reads. The development of accurate quality control methods is not only critical for fulfilling regulatory requirements, but will also provide novel insights into the biological mechanism of rAAV assembly in insect cells. This movie has been recorded by ICGEB Trieste.

Kas sina esindad enda tööandjat sotsiaalmeedias? Podcasti viib läbi 7Blaze juhataja Jaan Kruusma. Külalisteks on Ivar Raav, kes on Swedbank HR osakonnas projektijuht ning Kristel Leif, kelle meisterlikkuse läbi sünnib Swedbanki sotsiaalmeedia.

We assessed whether Angiopoietin-2 (Ang2), a Tie2 ligand and partial antagonist of Angiopoietin-1 (Ang1), is required for early vessel destabilization during postischemic angiogenesis, when combined with vascular growth factors. In vitro, matrigel co-cultures assessed endothelial-cell tube formation and pericyte recruitment after stimulation of VEGF-A, Apelin (APLN), Ang1 with or without Ang2. In a murine hindlimb ischemia model, adeno-associated virus (rAAV, 3×10(12) virusparticles) transduction of VEGF-A, APLN and Ang1 with or without Ang2 (continuous or early expression d0-3) was performed intramuscularly (d-14). Femoral artery ligation was performed at d0, followed by laser doppler perfusion meassurements (LDI) 7 and 14. At d7 (early timepoint) and d14 (late timepoint), histological analysis of capillary/muscle fiber ratio (CMF-R, PECAM-1) and pericyte/capillary ratio (PC-R, NG2) was performed. In vitro, VEGF-A, APLN and Ang1 induced ring formation, but only APLN and Ang1 recruited pericytes. Ang2 did not affect tube formation by APLN, but reduced pericyte recruitment after APLN or Ang1 overexpression. In vivo, rAAV.VEGF-A did not alter LDI-perfusion at d14, consistent with an impaired PC-R despite a rise in CMF-R. rAAV.APLN improved perfusion at d14, with or without continuous Ang2, increasing CMF-R and PC-R. rAAV.Ang1 improved perfusion at d14, when combined with rAAV.Ang2 (d0-3), accompanied by an increased CMF-R and PC-R. The combination of early vessel destabilization (Ang2 d0-3) and continuous Ang1 overexpression improves hindlimb perfusion, pointing to the importance of early vessel destabilization and subsequent vessel maturation for enhanced therapeutic neovascularization.

Prions are unconventional pathogens that cause transmissible spongiform encephalopathies (TSEs). According to the "protein only" hypothesis, prions consist of an infectious protein that is capable of converting a normal host protein termed PrPc into a protease resistant form termed PrPSc. PrPSc is poorly degraded by the host and accumulates in the CNS. Normal biological functions of PrPc and mechanisms involved in neurodegeneration remain obscure. During the past two decades, considerable efforts have been made to elucidate prion diseases and in particular to identify PrP interactors for a better understanding in prion biology. A major break-through was the identification of the 37 kDa laminin receptor (LRP), which represents the precursor of the human 67 kDa high-affinity laminin receptor (LR), as the cell surface receptor for the cellular prion protein. We investigated the role of LRP/LR in the propagation of PrPSc in chronically infected cells by different approaches. Three strategies resulted in downregulation or blocking of LRP and prevented PrPSc accumulation in different scrapie infected neuronal cell lines (i) transfection with an antisense LRP RNA expression plasmid (ii) transfection with small interfering (siRNAs) specific for the LRP mRNA and (iii) incubation with the polyclonal anti-LRP antibody, W3. We observed that the treatment with W3 abolished PrPSc deposition and reduced PrPc levels after one week of incubation. PrPSc did not reappear in cells being cultured for 14 additional days without therapeutic antibody treatment. Taken together, these results indicate that LRP is not only required for PrPc metabolism under non-pathological conditions but also has a pivotal role in prion propagation in a cell culture model. LRP/LR appears then to be a promising potential target for the development of therapeutics for the treatment of prion disease. Due to these encouraging cell culture data, we decided to select single chain antibodies (scFv) encompassing a suitable format for therapy. ScFvs are composed of variable parts of heavy and light chains of an immunoglobulin that are connected by a peptidic linker. The antibodies were screened on recombinant GST::LRP employing a phage display strategy. Two scFvs termed N3 and S18 were screened and selected by ELISA. Both antibodies were further characterized by western blotting and FACS analysis: both N3 and S18 specifically recognized mouseLRP and humanLRP overexpressed in mammalian cells under denaturating conditions (western blot) and under native conditions at the cell surface (FACS). Epitope mapping revealed that as expected both scFvs are directed against the extracellular part of LRP: S18 and N3 recognized amino acid residues 225-233 and 273-278, respectively. The ability of N3 and S18 to interfere with LRP/PrP interaction was tested by pull-down assays. In contrast to the control scFv C9 directed against the pre-S1 coat-protein of hepatitis B virus, both anti-LRP scFvs were able to block the specific LRP/PrP binding. In order to investigate a potential curing effect of scFv S18 in vivo, this scFv was tested in a scrapie mouse model by passive immunization. The application of S18 by intra-peritoneal injection was able to reduce PrPSc deposition in the spleen in comparison to mice injected with PBS or C9. However the survival times of S18 treated animals was not increased. Anti-LRP scFv S18 seems to contribute to block prion propagation in the periphery but it is likely that this effect was not enough strong to have an impact on the CNS invasion. Thus, we hypothesized that a strategy targeting directly the brain should be more effective. In this context, an approach based on the expression of single chain antibodies as secretory molecules in the brain via an adeno-associated virus (AAV) vector was initiated. To assure secretion of the scFv expressed in mammalian cells, a signal sequence was fused to the scFvs. Tranfection experiments demonstrated that neuronal cells were able to express and secrete high quantities of both scFvs. Furthermore, the generated scFvs were still functional as shown by western blotting. To find the appropriate AAV serotype for scFv expression, neuronal cells were transduced with varying serotypes carrying a GFP. AAV serotype 2 was chosen due to (i) its good transduction performance in two neuronal cell lines and (ii) the possibility of its purification by affinity chromatography. The sequences encoding for the scFvs N3, S18 and C9 have been cloned in an AAV-based vector. The AAV system was also able to drive high expression of scFvs into the supernatant by transfection or transduction. rAAV-scFv particles were produced and purifed for further stereotaxic injections into mice. Although the investigation of this therapeutic strategy is still in progress in a murine scrapie model, we already proved that a single injection of rAAV led to the expression of scFvs into the brain of mice 30 days post injection. This study represents the first gene therapeutic approach for the treatment of prion diseases.

Vectors based on adeno-associated virus type 2 (AAV) offer considerable promise for somatic gene therapy of various diseases (e.g. cystic fibrosis, hemophilia B, cancer). Limitations, however, still exist and require further improvement. The study presented here addresses two major problems that hamper a widespread use of AAV in human gene therapy: First, the loss of site-specific integration of recombinant AAV vectors (rAAV) due to the deletion of the rep gene, and secondly, the potential neutralization of AAV gene therapy vectors by preexisting antibodies. It could be demonstrated that site-specific integration of a transgene encoding rAAV vector can be efficiently restored by providing rep as plasmid DNA in trans. Based on these findings, a rAAV vector was developed where a plasmid coding for Rep was coupled as polylysine/DNA complex (PLL/DNA) directly to the capsid of the virion and co-transduction of such a PLL/DNA coupled to AAV was shown. Thus, providing rep-DNA as PLL/DNA should allow rAAV vectors to integrate specifically at chromosome 19. To characterize immunogenic domains of the AAV capsid and to develop strategies to circumvent neutralization by antibodies, AAV capsid mutants carrying peptide insertions in surface exposed loop regions were investigated in binding and neutralization assays. Three positions could be identified in the AAV capsid (at aa 534, 573, and 578 of the VP1 protein) where binding and neutralization of the virus by human serum samples was markedly reduced. These result suggest that capsid modifications could help to overcome binding and neutralization by human antisera in clinical gene therapy applications.