Altering the Ad5 Packaging Domain Affects the Maturation of the Ad Particles

We have previously described a new family of mutant adenoviruses carrying different combinations of attB/attP sequences from bacteriophage PhiC31 flanking the Ad5 packaging domain. These novel helper viruses have a significantly delayed viral life cycle and a severe packaging impairment, regardless of the presence of PhiC31 recombinase. Their infectious viral titers are significantly lower (100–1000 fold) than those of control adenovirus at 36 hours post-infection, but allow for efficient packaging of helper-dependent adenovirus. In the present work, we have analyzed which steps of the adenovirus life cycle are altered in attB-helper adenoviruses and investigated whether these viruses can provide the necessary viral proteins in trans. The entry of attB-adenoviral genomes into the cell nucleus early at early timepoints post-infection was not impaired and viral protein expression levels were found to be similar to those of control adenovirus. However, electron microscopy and capsid protein composition analyses revealed that attB-adenoviruses remain at an intermediate state of maturation 36 hours post-infection in comparison to control adenovirus which were fully mature and infective at this time point. Therefore, an additional 20–24 hours were found to be required for the appearance of mature attB-adenovirus. Interestingly, attB-adenovirus assembly and infectivity was restored by inserting a second packaging signal close to the right-end ITR, thus discarding the possibility that the attB-adenovirus genome was retained in a nuclear compartment deleterious for virus assembly. The present study may have substantive implications for helper-dependent adenovirus technology since helper attB-adenovirus allows for preferential packaging of helper-dependent adenovirus genomes

Long-term retinal PEDF overexpression prevents neovascularization in a murine adult model of retinopathy

Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders

Intravitreal neovessels in non-treated and AAV-null-treated 7.5 month-old TgIGF-I mice.

<p>(A) Representative image of PAS-stained sections of transgenic eyes showing intravitreal vessels. Note the presence of fibrotic tissue associated to the vessel attached to the inner limiting membrane of the retina, in an area where the retinal morphology appears altered. Wild-type eyes did not present vascular structures nor fibrosis in the vitreous cavity. Original magnification: 20× (B) ColIV (basal membrane marker) and CD105 (EC marker) inmunostaining in the same intravitreal neovessel depicted in (A) corresponding to a transgenic mouse aged 7.5 months. A clear CD105+ ring-shaped signal, indicating the presence of a lumen, was surrounded by Collagen IV+ staining. Arrows indicate completely formed intravitreal neovessels. Arrowheads indicate a blood vessel within the retina. Scale bar: 25 µm. (C) Representative image of the CD105+/Col-IV+ vascular structures <i>(asterisks)</i> present in the vitreous cavity of AAV2null-injected eyes 6 months after delivery of the vectors. No such structures were observed in the contralateral PEDF-treated eyes <i>(data not shown)</i>. The dashed line indicates the limit between the retina and the vitreous. Scale bar: 25 µm (<i>left pannel</i>), 7.5 µm (<i>right pannel</i>). <i>GCL</i>, ganglion cell layer; <i>L</i>, lenses, <i>V</i>, vitreous cavity, <i>R</i>, retina.</p

PEDF gene delivery prevents retinal detachment in TgIGF-I.

<p>Areas of retinal detachment were identified histologically by the presence of retinal folds. In most of the cases, infiltrating cells were observed in the subretinal space created around the detachment <i>(left panel, asterisks)</i>. Arrows indicate the monolayer of cells than constitute the Retinal Pigment Epithelium (RPE). Representative micrographs of the AAV-null and AAV2-hPEDF injected eyes of the same animal are shown. The incidence of retinal detachment was reduced from 83% to 16.5% after PEDF treatment (<i>n</i> = 6). <i>GCL</i>, ganglion cell layer; <i>INL</i>, inner nuclear layer; <i>ONL</i>, outer nuclear layer; <i>L</i>, lenses; <i>V</i>, vitreous; <i>SRS</i>, Subretinal space. Original magnification: 10×.</p

Specific cell types transduced by intravitreal AAV2 in TgIGF-I retinas.

<p>(A) Retinal flat-mount immunostained for GFP showing transduction of cells located on the surface of the retina. Arrows indicate GFP signal in axons, demonstrating transduction of ganglion cells. (B) GFP detection in paraffin-embedded eye sections. Besides transduction of cells in the ganglion cell layer (GCL), there was, to a lesser extend, transduction of cells of the Inner Nuclear layer (INL) <i>(inset, arrows)</i>. (C) Double immunostaining for GFP and calretinin (amacrine neurons) showed that amacrine cells located both in the surface of the retina as well as in the INL were transduced by AAV2-GFP. Bipolar cells, stained with an antibody against PKC-α, do not show colocalization with GFP nor in the cell bodies localized in the INL <i>(right inset)</i> nor in the neuronal projections ending at the GCL <i>(left inset)</i>. Immunohistochemistry for calbindin, expressed in horizontal cells of the INL outmost layer <i>(left inset)</i> and in a subtype of amacrine cells located in the INL and GCL <i>(right inset)</i>, showing that both cell types are transduced by AAV2-GFP. (D) Double immunostaining for GFP and Glutamine synthetase (Müller cells). No expression of GFP was observed in the Müller cells' end feet processes at the retinal surface <i>(right panel)</i> or in the cell bodies in the INL <i>(left panel)</i>. Astrocytes (S100+), localized in the most superficial retinal layer and around blood vessels, were not transduced by AAV2-GFP. Original magnification: 10× (A), 2× (B). Scale bar: 33 µm (C, <i>left and central panel</i>); 74 µm (C, <i>right panel</i>); 74 µm (D, <i>left panel</i>); 18 µm (D, <i>right panel</i>).</p

Inhibition of intraretinal neovascularization by AAV2-hPEDF treatment.

<p>(A) Representative images used for the quantitative assessment of intrarretinal capillary density. Original magnification: 40×. (B) Twenty high-magnification images of the capillary network that excluded major vessels were taken per retinal angiography with fluorescein-conjugated dextran, and the area corresponding to the capillary area was calculated using software tools as percentage of green area over total retinal area. Digital image quantification showed a significant increase in the retinal capillary density in transgenic animals compared with Wt. Six months after a single treatment with AAV2-hPEDF transgenic retinas showed a capillary density similar to that of Wt healthy retinas. N.I., non-injected. Values are expressed as the mean ±SEM of 7 animals/group. *<i>P</i><0.05.</p

Effects of AAV2-hPEDF treatment on VEGF levels in a HIF-1α independent manner.

<p>(A) Detection by ELISA of increased intraocular VEGF levels in both TgIGF-I and AAV2-null injected TgIGF-I mice. Levels normalized 6 months after AAV2-hPEDF treatment. (B) Time-course of retinal HIF-1α expression in TgIGF-I mice. HIF-1α protein was increased in retinas of 7.5 months-old TgIGF-I, remaining unaltered in younger animals. (C) AAV2-PEDF treatment did not affect HIF-1α expression 6 months after injection of the vector. N.I., non-injected. Values are expressed as the mean ±SEM of 4–10 animals/group. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05.</p

Long-term expression of hPEDF after single AAV2-mediated intravitreal gene delivery.

<p>(A) Time-course of hPEDF expression after a single intravitreal delivery of AAV2-hPEDF. Quantitative real time PCR analysis with transgene-specific primers revealed that hPEDF expression reached a plateau 2 months after a single injection of AAV2-hPEDF to 1.5-month-old TgIGF-I mice. hPEDF expression was undetectable in AAV2-null injected contralateral retinas. Values are expressed as the mean ±SEM of 3 animals/group. (B) Immunohistochemical detection of GFP <i>(left panel)</i> and PEDF <i>(right panel)</i> in retinal flat mounts 6 months after a single intravitreal injection of AAV2-GFP or AVV2-hPEDF demonstrating the expression of the exogenous transgenes. Whereas GFP is an intracellular protein that accumulates inside the cell, allowing the detection of the protein in neuronal dendrites and axons, PEDF is rapidly secreted after production, resulting in a more diffusse staining. Scale bar: 33 µm (C) PEDF Western Blot in retinas of transgenic treated mice. Wild-type, non-injected (N.I.) and AAV2-GFP-treated transgenic mice were used as controls. Values are expressed as the mean ±SEM of 4–6 animals/group. D) Immunodetection of human PEDF in AAV2- null <i>(left panel)</i> and AAV2-hPEDF <i>(right panel)</i> injected eyes verified that the PEDF overexpressed in ganglion cells was of vector-derived. Original magnification: 20×. (E) <i>Left panel</i>, inmunohistochemical detection of GFP (intracellular protein) in a flat-mounted retina 12 months after a single injection of AAV2-GFP to a wild-type mouse. <i>Right panels</i>, hPEDF (secreted protein) inmunodetection in paraffin-embedded eye sections 18 months after a single intravitreal injection of AAV2-hPEDF to TgIGF-I. Scale bars: 66 µm (<i>left panel</i>), 105 µm (<i>right upper panel</i>), 94 µm (<i>right lower panel</i>).</p

Production and neurotropism of lentivirus vectors pseudotyped with lyssavirus envelope glycoproteins.

International audienceWe investigated the production efficiency and the gene transfer capacity in the central nervous system of HIV-1-based vectors pseudotyped with either the G protein of the Mokola lyssaviruses (MK-G), a neurotropic virus causing rabies disease, or the vesiculo-stomatitis G protein (VSV-G). Both envelopes induced syncitia in cell cultures. They were incorporated into vector particles and mature virions were observed by electron microscopy. Vector production was two- to sixfold more efficient with VSV-G than with MK-G. For equivalent amounts of physical particles, vector titration was 5- to 25-fold higher with VSV-G than with MK-G pseudotypes on cultured cells, and in vivo gene expression in mouse brain was more intense. Thus, VSV-G pseudotypes were produced more efficiently and were more infectious than MK-G pseudotypes. Tropism for brain cells was analyzed by intrastriatal injections in rats. Both pseudotypes preferentially transduced neurons (70-90% of transduced cells). Retrograde axonal transport was investigated by instilling vector suspensions in the rat nasal cavity. Both pseudotypes were efficiently transported to olfactive neuron bodies. Thus, although coating HIV-1 particles with rabdhovirus envelope glycoproteins enables them to enter neuronal cells efficiently, pseudotyping is not sufficient to confer the powerful neurotropism of lyssaviruses to lentivirus vectors

Analysis of the excision and transposition processes.

<p>(<b>A</b>) Excision sites for the HSMAR2 transposase. Non-circled numbers correspond to the clone numbers, while encircled numbers correspond to the cleavage sites. (<b>B</b>) Transposition frequencies obtained with the mating-out assay (<b>C</b>) Amplification of the regions flanking the integration points. Gel bands (boxed) were cut and sequenced. DNA samples from the F plasmid from independent colonies of the mating-out (NalR + KanR + GentR) assays. 1: TT-clone, 2: TT-clone, 3: CG-clone, 4: CG-clone, 5: CG-clone. (<b>D</b>) Localization of the integration sites of the transposon ITR-Kan^R into the target plasmid pOX38. It shows the site of transposon insertion by analyzing the sequence obtained by BLAST. (<b>E</b>) Number of G418-resistant colonies after transposition in HeLa cells. The values correspond to the average of the neomycin-resistant clones of four independent experiments with n=3 per condition. It also shows the same results into two separate graphs according to whether the diameter of the cell clone if less than or equal to 1mm or is greater than 1mm. The asterisks indicate significant differences (the Student’s t-test, *p < 0,01; **p<0,001). Negative control (pCIneo) corresponds to a cotransfection of the Hsmar2 transposase with a plasmid carrying the Neo cassette but without the ITRs. In all cases, transfection conditions were 6 µg of plasmid per million cells.</p