Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models.

Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing.This study was funded by ProQR. iPSC work in the Cheetham lab is also supported by the Wellcome Trust, Fight for Sight, RP Fighting Blindness, and Moorfields Eye Charity

Antigen capsid-display on human adenovirus 35 via pIX fusion is a potent vaccine platform.

Durable protection against complex pathogens is likely to require immunity that comprises both humoral and cellular responses. While heterologous prime-boost regimens based on recombinant, replication-incompetent Adenoviral vectors (AdV) and adjuvanted protein have been able to induce high levels of concomitant humoral and cellular responses, complex manufacturing and handling in the field may limit their success. To combine the benefits of genetic and protein-based vaccination within one vaccine construct and to facilitate their use, we generated Human Adenovirus 35 (HAdV35) vectors genetically encoding a model antigen based on the Plasmodium falciparum (P. falciparum) circumsporozoite (CS) protein and displaying a truncated version of the same antigen (CSshort) via protein IX on the capsid, with or without a flexible glycine-linker and/or a 45Å-spacer. The four tested pIX-antigen display variants were efficiently incorporated and presented on the HAdV35 capsid irrespective of whether a transgene was encoded or not. Transgene-expression and producibility of the display-/expression vectors were not impeded by the pIX-display. In mice, the pIX-modified vectors induced strong humoral antigen-specific immunity that increased with the inclusion of the linker-/spacer molecules, exceeded the responses induced by the genetic, transgene-expressing HAdV35 vector, and surpassed recombinant protein in potency. In addition, the pIX- display/expression vectors elicited high antigen-specific cellular immune responses that matched those of the genetic HAdV35 vector expressing CS. pIX-modified display-/expression HAdV vectors may therefore be a valuable technology for the development of vaccines against complex pathogens, especially in resource-limited settings

Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models.

Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing.This study was funded by ProQR. iPSC work in the Cheetham lab is also supported by the Wellcome Trust, Fight for Sight, RP Fighting Blindness, and Moorfields Eye Charity

Immunogenicity of pIX-CS_short display is higher than that of CS protein and comparable levels cannot be achieved by mixing AdV vectors and protein.

<p>(A) Total CS-specific IgG titers in the serum of Balb/C mice 4 weeks post-immunization with 1x10¹⁰ VP/animal of HAdV35.empty.pIX-CS_short, HAdV35.CS.pIX-CS_short, with 5μg of CS protein in PBS, or with 1x10¹⁰ VP/animal HAdV35.empty. n = 7 per group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. (B) Total CS-specific IgG titers in the serum of Balb/C mice 4 weeks after immunization with 5μg CS protein in PBS, or mixed with 1x10¹⁰ VP/animal of empty AdV-vectors (HAdV35, HAdV26, or HAdV5), animals injected with Matrix-M only serve as control. n = 6 per group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. Ratio of CS-specific serum IgG2a to IgG1 levels, 4 weeks after immunization with 5μg CS protein in PBS only, or mixed with Matrix-M, or 1x10¹⁰ VP/animal of empty Ad-vectors (HAdV35, HAdV26, or HAdV5). Horizontal bars depict mean ratios (n = 5 animals/ group).</p

Characterization of the pIX-CS_short display- and display-/ expression vectors.

<p>(A) pIX-CS_short capsid incorporation in purified HAdV35 vector preparations. To confirm capsid incorporation of the pIX-CS_short (~50 kDa variants) 5 x10⁹ VP/well of each purified HAdV35 vector preparation was analyzed by Western blot using anti-CS antibody. To ensure equal loading, the blots were stained with anti-fiber 4D2 antibody (~35 kDa). The pIX-fusion proteins migrate higher than their predicted size in kDa due to the NANP-repeat in the CS protein. Marker (M) is indicated with the corresponding kDa band size. An additional band (~100 kDa) is indicated with an asterisk (*). (B) pIX-CS_short capsid display by electron microscopy (EM) anti-CS staining and gold-label staining. Representative EM images of HAdV35.empty.pIX-CS_short, HAdV35.CS.pIX-CS_short (upper row), HAdV35.empty.pIX-45-CS_short, HAdV35.CS.pIX-45-CS_short (middle row), HAdV35empty, and HAdV35 (bottom row) vectors. The bars represent 100 nm unless stated otherwise. (C) <i>In vitro</i> expression of CS transgene under non-replicating conditions in A549 cells. Western Blot analysis of cell lysates from A549 cells infected with 5000 VP/cell, using an anti-CS antibody. From left to right, the two controls HAdV35.empty and HAdV35.CS followed by the pIX-display vector variants with a CS transgene in E1 and without the transgene. Marker (M) is indicated with the corresponding kDa band size. (D) Heat Stability Assay showing percent (%) variation in luminescence in lysate of A549 cells infected with HAdV35.luc vector preparations subjected to 45°C temperature stress for 0 to 20 minutes. The percent variation was determined relative to the respective baseline time point (0 minutes).</p

pIX-CS_short display on CS-transgene expression-vectors induces strong humoral and cellular responses.

<p>(A) Total CS-specific IgG titers in the serum of Balb/C mice 2 and 8 weeks post-immunization with 1x10⁸, 1x10⁹, or 1x10¹⁰ VP/animal of HAdV35.empty.pIX-45-CS_short, HAdV35.CS.pIX-45-CS_short, a mix of HAdV35.empty.pIX-45-CS_short and HAdV35.CS, HAdV35.CS alone, or HAdV35.empty (10⁹ VP/ animal only). n = 5 per group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data by time point and dose. Black horizontal bars indicate statistical significance (p ≤ 0.05). (B) Ratio of CS-specific serum IgG2a to IgG1 levels, 4 weeks after immunization with 1x10¹⁰ VP of the HAdV35 vectors indicated in the legend. Horizontal bars depict mean ratios (n = 5 animals/ group). (C) IFNγ ELISPOT responses to stimulation of splenocytes of Balb/C mice 8 weeks post-immunization with <i>P</i>. <i>falciparum</i> CS peptide pool (left panel; “CS”) or the H-2Kd restricted immunodominant HAdV35 hexon epitope KYTPSNVTL (right panel; “HAdV35 hexon”). n = 5 animals/ group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. ns = not significant.</p

Design of HAdV35 pIX-display-vectors

<p>Schematic representation of pIX-display HAdV35 Advac vectors containing a human CMV promoter and SV40 poly-A expression cassette in E1 and the native pIX promoter (P). The vectors either express a 376 amino acid CS protein (lacking the GPI anchor) or no transgene (empty) in the E1 region. The pIX-display-vectors, with and without the CS-transgene in E1, are genetically modified to display the 151 amino acid CSshort with or without a 3 amino acid glycine-linker (Gly) and/or 45Å-spacer.</p