Susie E. Barker

Effective gene therapy with nonintegrating lentiviral vectors

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency–X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.

Long-term effect of gene therapy on Leber's congenital amaurosis.

BACKGROUND Mutations in RPE65 cause Lebers congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited. METHODS We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings. RESULTS Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG. CONCLUSIONS Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

The Drusenlike Phenotype in Aging Ccl2-Knockout Mice Is Caused by an Accelerated Accumulation of Swollen Autofluorescent Subretinal Macrophages

PURPOSE Drusen, which are defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2-knockout (Ccl2(-/-)) mice have been reported to develop drusen and phenotypic features similar to AMD, including an increased susceptibility to choroidal neovascularization (CNV). This study was conducted to investigate the nature of the drusenlike lesions in vivo and further evaluate the Ccl2(-/-) mouse as a model of AMD. METHODS The eyes of 2- to 25-month-old Ccl2(-/-) and C57Bl/6 mice were examined in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO) and electroretinography, and the extent of laser-induced CNV was measured by fluorescein fundus angiography. The retinal morphology was also assessed by immunohistochemistry and quantitative histologic and ultrastructural morphometry. RESULTS The drusenlike lesions of Ccl2(-/-) mice comprised accelerated accumulation of swollen CD68(+), F4/80(+) macrophages in the subretinal space that were apparent as autofluorescent foci on AF-SLO. These macrophages contained pigment granules and phagosomes with outer segment and lipofuscin inclusions that may account for their autofluorescence. Only age-related retinal pigment epithelium (RPE) damage, photoreceptor loss, and sub-RPE deposits were observed but, despite the accelerated accumulation of macrophages, we identified no spontaneous development of CNV in the senescent mice and found a reduced susceptibility to laser-induced CNV in the Ccl2(-/-) mice. CONCLUSIONS These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.

Assessment of ocular transduction using single-stranded and self-complementary recombinant adeno-associated virus serotype 2/8

To date adeno-associated viral (AAV) vectors are the only gene therapy vectors that have been shown to efficiently transduce photoreceptor cells and have thus become the most commonly used vector for ocular transduction. Various AAV serotypes have been evaluated in the eye, the first of which was AAV2, which is able to transduce photoreceptors, retinal pigment epithelium (RPE) and retinal ganglion cells. AAV serotypes 1 and 4, as well as AAV2 pseudotyped with these capsids, only transduce the RPE. AAV serotype 5 and AAV2/5 transduce the photoreceptors as well as RPE, but not retinal ganglion cells. Here, we assessed the capacity of the novel serotype AAV2/8 to transduce various ocular tissues of the adult murine retina by administering AAV2/8 green fluorescent protein intravitreally, subretinally and intracamerally. We also determined the kinetics and efficiency of self-complementary AAV (scAAV) vectors of serotypes 2/2, 2/5 and 2/8 and compared them with single-stranded AAV (ssAAV). We found that ssAAV2/8 transduces photoreceptors and RPE more efficiently than ssAAV2/2 and ssAAV2/5, and that scAAV2/8 had faster onset and higher transgene expression than ssAAV2/8. This improved transduction efficiency might facilitate the development of improved gene therapy protocols for inherited retinal degenerations, particularly those caused by defects in photoreceptor-specific genes.

Long-term survival of photoreceptors transplanted into the adult murine neural retina requires immune modulation.

Stem cell therapy presents an opportunity to replace photoreceptors that are lost as a result of inherited and age‐related degenerative disease. We have previously shown that murine postmitotic rod photoreceptor precursor cells, identified by expression of the rod‐specific transcription factor Nrl, are able to migrate into and integrate within the adult murine neural retina. However, their long‐term survival has yet to be determined. Here, we found that integrated Nrl.gfp+ve photoreceptors were present up to 12 months post‐transplantation, albeit in significantly reduced numbers. Surviving cells had rod‐like morphology, including inner/outer segments and spherule synapses. In a minority of eyes, we observed an early, marked reduction in integrated photoreceptors within 1 month post‐transplantation, which correlated with increased numbers of amoeboid macrophages, indicating acute loss of transplanted cells due to an inflammatory response. In the majority of transplants, similar numbers of integrated cells were observed between 1 and 2 months post‐transplantation. By 4 months, however, we observed a significant decrease in integrated cell survival. Macrophages and T cells were present around the transplantation site, indicating a chronic immune response. Immune suppression of recipients significantly increased transplanted photoreceptor survival, indicating that the loss observed in unsuppressed recipients resulted from T cell‐mediated host immune responses. Thus, if immune responses are modulated, correctly integrated transplanted photoreceptors can survive for extended periods of time in hosts with partially mismatched H‐2 haplotypes. These findings suggest that autologous donor cells are optimal for therapeutic approaches to repair the neural retina, though with immune suppression nonautologous donors may be effective. STEM CELLS 2010;28:1997–2007

EIAV vector-mediated delivery of endostatin or angiostatin inhibits angiogenesis and vascular hyperpermeability in experimental CNV

We evaluated the efficacy of equine infectious anaemia virus (EIAV)-based lentiviral vectors encoding endostatin (EIAV.endostatin) or angiostatin (EIAV.angiostatin) in inhibiting angiogenesis and vascular hyperpermeability in the laser-induced model of choroidal neovascularisation (CNV). Equine infectious anaemia virus.endostatin, EIAV.angiostatin or control (EIAV.null) vectors were administered into the subretinal space of C57Bl/6J mice. Two weeks after laser injury CNV areas and the degree of vascular hyperpermeability were measured by image analysis of in vivo fluorescein angiograms. Compared with EIAV.null-injected eyes, EIAV.endostatin resulted in a 59.5% (P<0.001) reduction in CNV area and a reduction in hyperpermeability of 25.6% (P<0.05). Equine infectious anaemia virus.angiostatin resulted in a 50.0% (P<0.05) reduction in CNV area and a 23.9% (P<0.05) reduction in hyperpermeability. Equine infectious anaemia virus.endostatin, but not EIAV.angiostatin significantly augmented the frequency of apoptosis within the induced CNV as compared with injected controls. TdT-dUTP terminal nick end labeling analysis 5 weeks post-injection, and histological and retinal flatmount analysis 12 months post-injection revealed no evidence of vector- or transgene expression-related deleterious effects on neurosensory retinal cells, or mature retinal vasculature in non-lasered eyes. Highly expressing EIAV-based vectors encoding endostatin or angiostatin effectively control angiogenesis and hyperpermeability in experimental CNV without long-term deleterious effects, supporting the use of such a strategy in the management of patients with exudative age-related macular degeneration.

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function.

The purpose of this study was to evaluate whether immune responses interfered with gene therapy rescue using subretinally delivered recombinant adeno-associated viral vector serotype 2 carrying the RPE65 cDNA gene driven by the human RPE65 promoter (rAAV2.hRPE65p.hRPE65) in the second eye of RPE65−/− dogs that had previously been treated in a similar manner in the other eye. Bilateral subretinal injection was performed in nine dogs with the second eye treated 85–180 days after the first. Electroretinography (ERG) and vision testing showed rescue in 16 of 18 treated eyes, with no significant difference between first and second treated eyes. A serum neutralizing antibody (NAb) response to rAAV2 was detected in all treated animals, but this did not prevent or reduce the effectiveness of rescue in the second treated eye. We conclude that successful rescue using subretinal rAAV2.hRPE65p.hRPE65 gene therapy in the second eye is not precluded by prior gene therapy in the contralateral eye of the RPE65−/− dog. This finding has important implications for the treatment of human LCA type II patients.

Subretinal delivery of adeno-associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice.

Adeno‐associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long‐term therapy is the development of immune responses against the vector or transgene product.

Lentiviral-vector-mediated expression of murine IL-1 receptor antagonist or IL-10 reduces the severity of endotoxin-induced uveitis

Uveitis is a sight threatening inflammatory disorder that remains a significant cause of visual loss. We investigated lentiviral gene delivery of interleukin 1 receptor antagonist (IL-1ra) or interleukin (IL)-10 to ameliorate murine endotoxin-induced uveitis (EIU). An human immunodeficiency virus-1-based vector containing the mIL-1ra or mIL-10 cDNA demonstrated high expression of biologically active cytokine. Following administration of Lenti.GFP into the anterior chamber, transgene expression was observed in corneal endothelial cells, trabecular meshwork and iris cells. To treat EIU, mice were injected with Lenti.IL-1ra, Lenti.IL-10 or a combination of these. EIU was induced 14 days after vector administration and mice were culled 12 h following disease induction. Lenti.IL-1ra or Lenti.IL-10-treated eyes showed significantly lower mean inflammatory cell counts in the anterior and posterior chambers compared with controls. The aqueous total protein content was also significantly lower in treated eyes, demonstrating better preservation of the blood-ocular barrier. Furthermore, the treated eyes showed less in vivo fluorescein leakage from inner retinal vessels compared with controls. The combination of both IL-1ra and IL-10 had no additive effect. Thus, lentiviral gene delivery of IL-1ra or IL-10 significantly reduces the severity of experimental uveitis, suggesting that lentiviral-mediated expression of immunomodulatory genes in the anterior chamber offers an opportunity to treat uveitis.

Targeted Gene Delivery to Human Airway Epithelial Cells with Synthetic Vectors Incorporating Novel Targeting Peptides Selected by Phage Display

Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including R/KSM, LP/QHK and PSGA/TARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.