Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates

Nathwani, Amit C.; Gray, John T.; McIntosh, Jenny; Ng, Catherine Y.; Zhou, Junfang; Spence, Yunyu; Cochrane, Melanie; Gray, Elaine; Tuddenham, Edward G. D.; Davidoff, Andrew M.

doi:10.1182/blood-2006-03-010181

Cited by 244 publications

(237 citation statements)

References 21 publications

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“…The second-generation optimized CsCl purification method described and characterized herein enables flexible purification of vectors of varying serotypes, and importantly results in consistently high vector purity and potency, and comparable in these attributes to vectors prepared for clinical studies. The use of such improved AAV purification methods for pre-clinical studies, combined with further improvements in expression cassettes, use of self-complementary AAV constructs, 23 more efficient serotypes, 8,24 and improved delivery techniques are important strategies to meet the need for high levels of therapeutic transgene expression and support successful translational research.…”

Section: Discussionmentioning

confidence: 99%

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

et al. 2009

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The purity of adeno-associated virus (AAV) vector preparations has important implications for both safety and efficacy of clinical gene transfer. Early-stage screening of candidates for AAV-based therapeutics ideally requires a purification method that is flexible and also provides vectors comparable in purity and potency to the prospective investigational product manufactured for clinical studies. The use of cesium chloride (CsCl) gradient-based protocols provides the flexibility for purification of different serotypes; however, a commonly used first-generation CsCl-based protocol was found to result in AAV vectors containing large amounts of protein and DNA impurities and low transduction efficiency in vitro and in vivo. Here, we describe and characterize an optimized, secondgeneration CsCl protocol that incorporates differential precipitation of AAV particles by polyethylene glycol, resulting in higher yield and markedly higher vector purity that correlated with better transduction efficiency observed with several AAV serotypes in multiple tissues and species. Vectors purified by the optimized CsCl protocol were found to be comparable in purity and functional activity to those prepared by more scalable, but less flexible serotype-specific purification processes developed for manufacture of clinical vectors, and are therefore ideally suited for pre-clinical studies supporting translational research.

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Section: Discussionmentioning

confidence: 99%

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

et al. 2009

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“…However, studies in animals and humans have suggested that the AAV capsid mounts a very strong TD immune response. The evidence includes the following: (i) no NAb was detected after muscular injection, and AAV vector transgene expression was observed following readministration of AAV2 vector via muscular injection in MHC class II-deficient mice (42); (ii) transient immunosuppression with CD4 antibody treatment at the time of primary infection elicited no NAbs and allowed transgene expression in wild-type mice following readministration of AAV vectors (19,42,66); (iii) our prior work has demonstrated that a high NAb titer was achieved after immunization with AAV-pulsed dendritic cells in mice (34); (iv) IgG subclasses were produced in mice and primates with AAV administration (19,52,53,69); and (v) IgG subclasses were observed in humans and in patients following AAV vector treatment (6,51,54). In this study, isotype assays suggested that IgG2a is the predominant IgG subclass in all mice immunized with AAV1 to -5, which is consistent with other viruses that also elicit markedly increased IgG2a production in mice (20).…”

Section: Discussionmentioning

confidence: 99%

Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

DiPrimio

Bowles

et al. 2012

J Virol

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Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration.A deno-associated virus (AAV) vectors have been safely and successfully used in phase I clinical trials (39,40,54). In particular, therapeutic effects have been achieved in patients with Leber's congenital amaurosis and hemophilia B. Among 30 patients with Leber's congenital amaurosis, 28 demonstrated remarkably improved eyesight after AAV type 2 (AAV2) delivery of the rpe65 gene to the retina. Regarding the recent hemophilia B trial, all six patients had therapeutic factor IX (FIX) expression within a beneficial 1% to 8% of normal levels as long as 18 months after intravenous delivery of an AAV vector encoding an optimized FIX cassette (54).AAV is a nonenveloped, single-stranded DNA virus that requires a helper virus, such as adenovirus (Ad) or herpes simplex virus, for efficient replication. Despite the lack of inflammation or other AAV-associated complications following administration of AAV2 vectors in several organs, neutralizing antibody (NAb) titers against the AAV2 capsid were found to be significantly increased following vector administration, particularly in the lung, muscle, and liver (7, 8, 43-45, 49, 54, 63). NAbs in circulation are able to block AAV transduction after systemic administration. Recently, Manno et al. (44) performed a phase I study of AAV-mediated FIX transgene delivery in patients with hemophilia B, in which one patient with a higher preexisting NAb ti...

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“…The same was shown in non-human primates using AAV5. 74 Intravenous administration of a self-complementary AAV5 vector encoding the human F.IX gene resulted in comparable levels of circulating F.IX in macaques with or without pre-existing immunity to AAV8. This confirms that alternative serotypes can circumvent pre-existing naturally acquired immunity to AAV in the non-human primate model.…”

Section: Aav Capsid Antibodies: a Persistent Challengementioning

confidence: 99%

Immunity to adeno-associated virus vectors in animals and humans: a continued challenge

2008

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Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates

Cited by 244 publications

References 21 publications

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

Immunity to adeno-associated virus vectors in animals and humans: a continued challenge

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