Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.

Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.

MCARD-mediated gene transfer of GRK2 inhibitor in ovine model of acute myocardial infarction.

ß-Adrenergic receptor (ßAR) dysfunction in acute myocardial infarction (MI) is associated with elevated levels of the G-protein-coupled receptor kinase-2 (GRK2), which plays a key role in heart failure progression. Inhibition of GRK2 via expression of a peptide ßARKct transferred by molecular cardiac surgery with recirculating delivery (MCARD) may be a promising intervention. Five sheep underwent scAAV6-mediated MCARD delivery of ßARKct, and five received no treatment (control). After a 3-week period, the branch of the circumflex artery (OM1) was ligated. Quantitative PCR data showed intense ßARKct expression in the left ventricle (LV). Circumferential fractional shortening was 23.4 ± 7.1 % (baseline) vs. -2.9 ± 5.2 % (p < 0.05) in the control at 10 weeks. In the MCARD-ßARKct group, this parameter was close to baseline. The same trend was observed with LV wall thickening. Cardiac index fully recovered in the MCARD-ßARKct group. LV end-diastolic volume and LV end-diastolic pressure did not differ in both groups. MCARD-mediated ßARKct gene expression results in preservation of regional and global systolic function after acute MI without arresting progressive ventricular remodeling.

A pharmacokinetic analysis of molecular cardiac surgery with recirculation mediated delivery of ßARKct gene therapy: developing a quantitative definition of the therapeutic window.

BACKGROUND: Two major problems for translating gene therapy for heart failure therapy are: safe and efficient delivery and the inability to establish a relationship between vector exposure and in vivo effects. We present a pharmacokinetics (PK) analysis of molecular cardiac surgery with recirculating delivery (MCARD) of scAAV6-ßARKct. MCARD's stable cardiac specific delivery profile was exploited to determine vector exposure, half-life, and systemic clearance. METHODS AND RESULTS: Five naive sheep underwent MCARD with 10(14) genome copies of scAAV6-ßARKct. Blood samples were collected over the recirculation interval time of 20 minutes and evaluated with quantitative polymerase chain reaction (qPCR). C(t) curves were generated and expressed on a log scale. The exposure, half-life, and clearance curves were generated for analysis. qPCR and Western blots were used to determine biodistribution. Finally, all in vivo transduction data was plotted against MCARD's PK to determine if a relationship existed. Vector concentrations at each time point were (cardiac and systemic, respectively): 5 minutes: 9.16 ± 0.15 and 3.21 ± 0.38; 10 minutes: 8.81 ± 0.19 and 3.62 ± 0.37; 15 minutes: 8.75 ± 0.12 and 3.69 ± 0.31; and 20 minutes: 8.66 ± 0.22 and 3.95 ± 0.26; P < .00001. The half life of the vector was 2.66 ± 0.24 minutes. PK model data revealed that only 0.61 ± 0.43% of the original dose remained in the blood after delivery, and complete clearance from the system was achieved at 1 week. A PK transfer function revealed a positive correlation between exposure and in vivo transduction. Robust ßARKct expression was found in all cardiac regions with none in the liver. CONCLUSION: MCARD may offer a viable method to establish a relationship between vector exposure and in vivo transduction. Using this methodology, it may be possible to address a critical need for establishing an effective therapeutic window.

The poly(rC)-binding protein alphaCP2 is a noncanonical factor in X. laevis cytoplasmic polyadenylation.

Post-transcriptional control of mRNA stability and translation is central to multiple developmental pathways. This control can be linked to cytoplasmic polyadenylation in certain settings. In maturing Xenopus oocytes, specific mRNAs are targeted for polyadenylation via recruitment of the Cytoplasmic Polyadenylation Element (CPE) binding protein (CPEB) to CPE(s) within the 3' UTR. Cytoplasmic polyadenylation is also critical to early embryonic events, although corresponding determinants are less defined. Here, we demonstrate that the Xenopus ortholog of the poly(rC) binding protein αCP2 can recruit cytoplasmic poly(A) polymerase activity to mRNAs in Xenopus post-fertilization embryos, and that this recruitment relies on cis sequences recognized by αCP2. We find that the hα-globin 3' UTR, a validated mammalian αCP2 target, constitutes an effective target for cytoplasmic polyadenylation in Xenopus embryos, but not during Xenopus oocyte maturation. We further demonstrate that the cytoplasmic polyadenylation activity is dependent on the action of the C-rich αCP-binding site in conjunction with the adjacent AAUAAA. Consistent with its ability to target mRNA for poly(A) addition, we find that XαCP2 associates with core components of the Xenopus cytoplasmic polyadenylation complex, including the cytoplasmic poly(A) polymerase XGLD2. Furthermore, we observe that the C-rich αCP-binding site can robustly enhance the activity of a weak canonical oocyte maturation CPE in early embryos, possibly via a direct interaction between XαCP2 and CPEB1. These studies establish XαCP2 as a novel cytoplasmic polyadenylation trans factor, indicate that C-rich sequences can function as noncanonical cytoplasmic polyadenylation elements, and expand our understanding of the complexities underlying cytoplasmic polyadenylation in specific developmental settings.

Current strategies for myocardial gene delivery.

Existing methods of cardiac gene delivery can be classified by the site of injection, interventional approach and type of cardiac circulation at the time of transfer. General criteria to assess the efficacy of a given delivery method include: global versus regional myocardial transduction, technical complexity and the pathophysiological effects associated with its use, delivery-related collateral expression and the delivery-associated inflammatory and immune response. Direct gene delivery (intramyocardial, endocardial, epicardial) may be useful for therapeutic angiogenesis and for focal arrhythmia therapy but with gene expression which is primarily limited to regions in close proximity to the injection site. An often unappreciated limitation of these techniques is that they are frequently associated with substantial systemic vector delivery. Percutaneous infusion of vector into the coronary arteries is minimally invasive and allows for transgene delivery to the whole myocardium. Unfortunately, efficiency of intracoronary delivery is highly variable and the short residence time of vector within the coronary circulation and significant collateral organ expression limit its clinical potential. Surgical techniques, including the incorporation of cardiopulmonary bypass with isolated cardiac recirculation, represent novel delivery strategies that may potentially overcome these limitations; yet, these techniques are complex with inherent morbidity that must be thoroughly evaluated before safe translation into clinical practice. Characteristics of the optimal technique for gene delivery include low morbidity, increased myocardial transcapillary gradient, extended vector residence time in the coronary circulation and exclusion of residual vector from the systemic circulation after delivery to minimize extracardiac expression and to mitigate a cellular immune response. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy".

Effects of local mRNA structure on posttranscriptional gene silencing.

Antisense oligodeoxynucleotides (AONs) and short interfering RNAs (siRNAs) effect posttranscriptional gene silencing (PTGS) by hybridizing to an mRNA and then directing its cleavage. To understand the constraints that mRNA structure imposes on AON- vs. siRNA-mediated PTGS, AON- and siRNA-mediated cleavage of defined mRNA structures was monitored in Drosophila embryo whole-cell lysates. We observed that AON-directed cleavage was approximately 3-fold faster than cleavage with a siRNA directed to the same target site. Furthermore, and unexpectedly, AON-mediated cleavage was found to be much less fastidious with respect to target sequence accessibility, as measured by the presence of unpaired nucleotides, than a corresponding siRNA. Nonetheless, in vivo, siRNAs silenced their mRNA target at least 2-fold more efficiently than the corresponding AON. These seemingly contradictory results suggested that additional, as yet undefined factors play an important role in regulating PTGS efficiency in vivo. We used a well defined RNA-binding protein, alphaCP, and its corresponding high-affinity RNA-binding site to explore this hypothesis. We found that prebound alphaCP effectively blocked AON-mediated cleavage of the RNA-binding site compared with cleavage of the site in the absence of alphaCP. We conclude that higher-order structures formed by RNA and bound proteins play an important role in determining the efficiency of AON-directed PTGS. We hypothesize that strategies aimed at removing RNA-binding proteins might significantly improve AON-mediated PTGS in vivo.

Shared stabilization functions of pyrimidine-rich determinants in the erythroid 15-lipoxygenase and alpha-globin mRNAs.

The poly(C)-binding proteins, alphaCPs, comprise a set of highly conserved KH-domain factors that participate in mRNA stabilization and translational controls in developmental and viral systems. Two prominent models of alphaCP function link these controls to late stages of erythroid differentiation: translational silencing of 15-lipoxygenase (Lox) mRNA and stabilization of alpha-globin mRNA. These two controls are mediated via association of alphaCPs with structurally related C-rich 3'-untranslated region elements: the differentiation control elements (DICE) in Lox mRNA and the pyrimidine-rich motifs in alpha-globin mRNA. In the present report a set of mRNA translation and stability assays are used to determine how these two alphaCP-containing complexes, related in structure and position, mediate distinct posttranscriptional controls. While the previously reported translational silencing by the DICE is not evident in our studies, we find that the two determinants mediate similar levels of mRNA stabilization in erythroid cells. In both cases this stabilization is sensitive to interference by a nuclear-restricted alphaCP decoy but not by the same decoy restricted to the cytoplasm. These data support a general role for alphaCPs in stabilizing a subset of erythroid mRNAs. The findings also suggest that initial binding of alphaCP to target mRNAs occurs in the nucleus. Assembly of stabilizing mRNP complexes in the nucleus prior to export may maximize their impact on cytoplasmic events.

Soluble HIV-specific T cell receptor: expression, purification and analysis of the specificity.

We have produced soluble T cell receptor (TCR) derived from a human CD8(+) cytotoxic T lymphocyte (CTL) clone D3 that recognizes the immunodominant HIV Gag peptide SLYNTVATL (SL9) in association with major histocompatibility complex (MHC) class I protein HLA-A2. Drosophila Schneider cells (S2) were used to express genes coding the TCR alpha and beta chains under an inducible promoter. Both chains were labeled with two different tags: a (His)(6) was introduced at the C-terminal end of alpha chain, while beta chain was terminated by c-myc. Since an isolated alpha chain is unstable unless it is associated with a beta chain, this design permits rapid separation of alpha,beta-heterodimer from unpaired beta chain in a single step of Ni-NTA Agarose chromatography yielding 90% pure alpha,beta-TCR. Introduction of the c-myc epitope to the beta chain allows capture of soluble D3 from the culture supernatant by immobilized anti-c-myc antibody, without the need for receptor purification. The TCR specificity was then examined by analyzing the binding of peptide-HLA-A2/tetramer in an ELISA assay. Using this assay, we have also evaluated the binding of monomeric SL9-HLA-A2 complex to the immobilized D3 TCR and determined that the affinity measurement of the D3-SL9-HLA-A2 reaction is similar to that obtained by a biosensor instrument. We propose that the approach described here is generally useful for purification of other soluble TCRs and will allow rapid analysis of their specificity.