WO2016037191A1 - Utilisation de plasmides et peptides dérivés de l'huntingtine pour une immunisation active comme agent thérapeutique de la maladie de huntington (hd) - Google Patents

Utilisation de plasmides et peptides dérivés de l'huntingtine pour une immunisation active comme agent thérapeutique de la maladie de huntington (hd) Download PDF

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WO2016037191A1
WO2016037191A1 PCT/US2015/049025 US2015049025W WO2016037191A1 WO 2016037191 A1 WO2016037191 A1 WO 2016037191A1 US 2015049025 W US2015049025 W US 2015049025W WO 2016037191 A1 WO2016037191 A1 WO 2016037191A1
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residues
combination
plasmid
mice
polyq
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Anne Messer
Arlene RAMSINGH
Kevin Manley
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Health Research, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

  • This present invention relates to, inter alia, therapeutics and therapeutic methods for treating Huntington's disease (HD).
  • HD Huntington's disease
  • Huntington's disease The identity of the Huntington's disease (HD) gene and the general concept of the misfolding protein that triggers pathogenesis have been known for many years. Further, Huntington's disease has been a paradigm disease for neurodegenerations triggered by misfolding proteins, since it has been possible to develop cellular and animal models with robust readouts. However, effective protective therapies for Huntington's disease have not yet been developed.
  • the present invention relates to, inter alia, therapeutics and therapeutic methods for treating Huntington's disease (HD).
  • HD Huntington's disease
  • the present invention relates to use of Huntingtin-derived plasmids and peptides for active immunization as a Huntington's disease therapeutic.
  • the present invention relates to a therapeutic for treating
  • Huntington's disease said therapeutic comprising an immunogen that can induce protective immunity against the HD phenotype.
  • the present invention relates to a method of treating
  • Huntington's disease said method comprising administering to a subject in need of HD treatment a therapeutic comprising an immunogen that can induce protective immunity against the HD phenotype.
  • Figure 1 is a graph illustrating mutant HTT concentrations (fmol/mg total protein) detected in Het ZQ175 mouse brain.
  • Figures 2A-2B are graphs illustrating antibody responses obtained with TriPep and N586-Q82 plasmid in fragment model R6/1 ( Figure 2A) and full length knock-in model zQ175 ( Figure 2B).
  • Figures 3A-3B are Western blot results relating to plasmid-vaccinated mice screened for antibody against the encoded protein.
  • Total cell lysate from transfected HEK 293T cells was used for SDS-PAGE. W markers (kDa) are shown on the left.
  • Anti-GFP ( Figure 3A) and anti-GST ( Figure 3B) monoclonal antibodies were used as positive controls, and mouse serum was used at 1 : 100 dilution to probe for the plasmid-encoded proteins.
  • GFP, HD25Q-GFP, and HD103Q-GFP Figure 3 A
  • HD103QGST and DRPLA62Q-GST Figure 3B were used to detect huntingtin- and poly-Qspecific antibodies, respectively.
  • Figures 4A-4C illustrate that immune response to HD plasmid vaccination is correlated directly with amelioration of the HDR6/2 diabetic phenotype.
  • Figure 4A At 11 weeks, pHD103QGFP-immunoresponsive HDR6/2 mice had improved 6-h fasting blood glucose compared to HDR6/2 controls. Statistical comparisons indicated are against HDR6/2 pHD103Q-GFP-immunoresponsive group.
  • Figure 4B Untreated HDR6/2 mice had significantly higher overnight fasting blood glucose than DNA-vaccinated HDR6/2 groups. Statistical comparisons indicated are against wild-type group.
  • Figure 4C Glucose tolerance testing revealed a more penetrant diabetic phenotype.
  • Glucose challenge demonstrated that pHD103Q-GFP-immunoresponsive HDR6/2 mice had lower blood glucose than untreated and control-treated HDR6/2 mice.
  • Statistical comparisons indicated are against wild-type group. WT, wild type; HD103 resp, HDR6/2 pHD103Q-GFP immunoresponsive; HD103Q- GFP non-resp, HDR6/2 pHD103Q-GFP nonimmunoresponsive; HDpGFP, HDR6/2 pGFP- treated; HD17AA, HDR6/2 17AA peptide-treated; HD CpG 1639, 3Db, CpGoligonucleotide DNA-treated; HD UT, HDR6/2 untreated.
  • #P 0.053; *P ⁇ 0.05; **P ⁇ 0.01 (statistical analysis was calculated by Mann-Whitney U test between groups).
  • Figures 5A-5D illustrate immune response to HD plasmid vaccination protects against pancreatic insulin dysfunction in HDR6/2 mice. Mice were perfused at 12.5-13 weeks of age, and pancreas was harvested and paraffin-embedded. Five-micrometer slices were immunostained for insulin, and images were exposure-matched. Wild-type islets (Figure 5A) show strong, homogeneous insulin staining, while islets in untreated HDR6/2 mice ( Figure 5B) show an overall decrease in insulin, with few strongly positive areas.
  • pHD103Q-GFP-immunoresponsive HDR6/2 mice show insulin recovery more closely resembling wild-type islets; islets of nonresponsive HDR6/2 mice (Figure 5D) exhibited no changes in insulin staining relative to untreated HDR6/2 controls. All HDR6/2 mice displayed cytoplasmic shrinkage of islet cells. Scale bar, 30 m.
  • Figure 6 is a graph relating to studies showing a vaccine immunogen consisting of three non-overlapping HTT exon 1 peptides induces similar antibody titers in immunized HD mutant and wild-type mice.
  • Serum antibody titers (reciprocal of the serum dilution whose absorbance is 3X that of negative controls) were determined by ELISA after immunization of zQ175, R6/1, or wild-type mice with a triple peptide combination, the N586 plasmid DNA, or a prime/boost regimen ( 586 prime, triple peptide boost).
  • One-way ANOVA was used to identify differences among the three vaccine immunogens or the three mouse strains.
  • Figures 7A-7B illustrate GSEA output for one gene set, Biocarta IL22BP.
  • Figure 7A Enrichment score reflecting the degree to which genes in Biocarta IL22BP are overrepresented at the top of the ranked list of genes in a comparison of immunized R6/1 versus unimmunized R6/1 mice.
  • Figure 7B Ranked gene expression data displayed as a heat map for four immunized R6/ 1 mice (I) versus five unimmunized R6/ 1 mice (UI). The leading-edge subset contains four probes (SOCS3, SOCS3, IL22RA2, and IL22) and is delineated by a vertical line.
  • This present invention relates to, inter alia, therapeutics and therapeutic methods for treating Huntington's disease (HD).
  • the present invention relates to use of Huntingtin-derived plasmids and peptides for active immunization as a Huntington's disease (HD) therapeutic.
  • the present invention relates to a therapeutic for treating
  • Huntington's disease said therapeutic comprising an immunogen that can induce protective immunity against the HD phenotype.
  • the immunogen is as disclosed herein.
  • the immunogen comprises a peptide of human HTT protein.
  • the peptide of human HTT protein is selected from the group consisting of N17 (first 17 residues of exonl), PP (residues 49-60), and EM48 (residues 74-88).
  • the immunogen comprises a combination of peptides of human HTT protein.
  • the combination of peptides of human HTT protein comprises at least one of the peptides selected from the group consisting of N17 (first 17 residues of exonl), PP (residues 49-60), and EM48 (residues 74-88).
  • each peptide of the combination is chemically linked to hapten keyhole limpet hemocynanin (KLH).
  • the combination of peptides of human HTT protein comprises N17 (first 17 residues of exonl), PP (residues 49-60), and EM48 (residues 74-88).
  • each peptide of the combination is chemically linked to hapten keyhole limpet hemocynanin (KLH).
  • KLH hapten keyhole limpet hemocynanin
  • the immunogen comprises a plasmid comprising nucleotides encoding the AAs of human HTT exonl, with 97 polyglutamines (polyQ), wherein the polyQ region is encoded by a combination of CAG and CAA nucleotides to prevent the instability deriving from an uninterrupted tract of CAG repeats.
  • the immunogen comprises a plasmid comprising nucleotides encoding the AAs of human HTT exonl, with 97 polyglutamines (polyQ), wherein the polyQ region is encoded by a combination of CAG and CAA nucleotides to prevent the instability deriving from an uninterrupted tract of CAG repeats, and optionally with adjuvants such as fused sequences encoding small peptides known to increase non-specific T-helper cell activity, and optionally being co-administered with specific cytokine cDNAs.
  • polyQ polyglutamines
  • the immunogen comprises a plasmid comprising nucleotides encoding the first 586AAs of human HTT, with 82 polyglutamines (polyQ), wherein the polyQ region is encoded by a combination of CAG and CAA nucleotides to prevent the instability deriving from an uninterrupted tract of CAG repeats, and optionally with adjuvants such as fused sequences encoding small peptides known to increase non-specific T-helper cell activity, and optionally being co-administered with specific cytokine cDNAs.
  • polyQ polyglutamines
  • the immunogen comprises a combination of at least one peptide of human HTT protein and at least one plasmid selected from the group consisting of: (a) a plasmid comprising nucleotides encoding the AAs of human HTT exonl, with 97 polyglutamines (polyQ), wherein the polyQ region is encoded by a combination of CAG and CAA nucleotides to prevent the instability deriving from an uninterrupted tract of CAG repeats; (b) a plasmid comprising nucleotides encoding the AAs of human HTT exonl, with 97 polyglutamines (polyQ), wherein the polyQ region is encoded by a combination of CAG and CAA nucleotides to prevent the instability deriving from an uninterrupted tract of CAG repeats, and optionally with adjuvants such as fused sequences encoding small peptides known to increase non-specific T-helper cell activity,
  • the at least one peptide of human HTT protein is selected from the group consisting of N17 (first 17 residues of exonl), PP (residues 49-60), and EM48 (residues 74-88).
  • the combination of at least one peptide of human HTT protein comprises N17 (first 17 residues of exonl), PP (residues 49-60), and EM48 (residues 74-88).
  • each peptide of the combination is chemically linked to hapten keyhole limpet hemocynanin (KLH).
  • KLH hapten keyhole limpet hemocynanin
  • the immunogen comprises a plasmid that may be delivered via particle mediated epidermal delivery (PMED), gene gun, electroporation, and/or a viral vector (e.g., AAV; Coxsackie B).
  • PMED particle mediated epidermal delivery
  • AAV AAV
  • Coxsackie B a viral vector
  • the present invention relates to a method of treating
  • Huntington's disease said method comprising administering to a subject in need of HD treatment a therapeutic comprising an immunogen that can induce protective immunity against the HD phenotype.
  • the immunogen is as disclosed herein.
  • the immunogen comprises a peptide of human HTT protein.
  • the peptide of human HTT protein is selected from the group consisting of N17 (first 17 residues of exonl), PP (residues 49-60), and EM48 (residues 74-88).
  • the immunogen comprises a combination of peptides of human HTT protein.
  • the combination of peptides of human HTT protein comprises at least one of the peptides selected from the group consisting of N17 (first 17 residues of exonl), PP (residues 49-60), and EM48 (residues 74- 88).
  • each peptide of the combination is chemically linked to hapten keyhole limpet hemocynanin (KLH).
  • KLH hapten keyhole limpet hemocynanin
  • the combination of peptides of human HTT protein comprises N17 (first 17 residues of exonl), PP (residues 49- 60), and EM48 (residues 74-88).
  • each peptide of the combination is chemically linked to hapten keyhole limpet hemocynanin (KLH).
  • KLH hapten keyhole limpet hemocynanin
  • the immunogen comprises a plasmid comprising nucleotides encoding the AAs of human HTT exonl, with
  • polyQ polyglutamines
  • the immunogen comprises a plasmid comprising nucleotides encoding the AAs of human HTT exonl, with 97 polyglutamines (polyQ), wherein the polyQ region is encoded by a combination of CAG and CAA nucleotides to prevent the instability deriving from an uninterrupted tract of CAG repeats, and optionally with adjuvants such as fused sequences encoding small peptides known to increase non-specific T-helper cell activity, and optionally being co-administered with specific cytokine cDNAs.
  • polyQ polyglutamines
  • the immunogen comprises a plasmid comprising nucleotides encoding the first 586AAs of human HTT, with 82 polyglutamines (polyQ), wherein the polyQ region is encoded by a combination of CAG and CAA nucleotides to prevent the instability deriving from an uninterrupted tract of CAG repeats, and optionally with adjuvants such as fused sequences encoding small peptides known to increase non-specific T-helper cell activity, and optionally being co-administered with specific cytokine cDNAs.
  • polyQ polyglutamines
  • the immunogen comprises a combination of at least one peptide of human HTT protein and at least one plasmid selected from the group consisting of: (a) a plasmid comprising nucleotides encoding the AAs of human HTT exonl, with 97 polyglutamines (polyQ), wherein the polyQ region is encoded by a combination of CAG and CAA nucleotides to prevent the instability deriving from an uninterrupted tract of CAG repeats; (b) a plasmid comprising nucleotides encoding the AAs of human HTT exonl, with 97 polyglutamines (polyQ), wherein the polyQ region is encoded by a combination of CAG and CAA nucleotides to prevent the instability deriving from an uninterrupted tract of CAG repeats, and optionally with adjuvants such as fused sequences encoding small peptides known to increase non-specific T-helper cell activity,
  • the at least one peptide of human HTT protein is selected from the group consisting of N17 (first 17 residues of exonl), PP (residues 49-60), and EM48 (residues 74-88).
  • the combination of at least one peptide of human HTT protein comprises N17 (first 17 residues of exonl), PP (residues 49-60), and EM48 (residues 74-88).
  • each peptide of the combination is chemically linked to hapten keyhole limpet hemocynanin (KLH).
  • KLH hapten keyhole limpet hemocynanin
  • the immunogen comprises a plasmid that is delivered via particle mediated epidermal delivery (PMED), gene gun, electroporation, and/or a viral vector (e.g., AAV; Coxsackie B).
  • PMED particle mediated epidermal delivery
  • AAV AAV
  • Coxsackie B a viral vector
  • Immunogens will consist of N586-82Q plasmid and Htt exonl KLH conjugated peptides representing known epitopes (1-17; 49-60; 74-88), at doses determined in our current project.
  • Five test groups will consist of plasmid, peptides, plasmid+peptides; negative plasmid control; mock immunized control.
  • Pharmacokinetics (PK) will include ELISA measurements of anti-HTT antibody levels in serum and tissues.
  • Pharmacodynamic (PD) behavioral readouts will include both motor and activity (roto-rod and open-field activity) and cognitive (Two-Choice swim maze) behavioral tasks.
  • Molecular readouts are gene transcriptional profiles of brain and peripheral blood mononuclear cells (PBMCs). Additional tissues will be archived for future studies.
  • H7Texonl-Q97 or N586-82Q (encoding the first 586 residues of the HTT) were delivered via particle mediated epidermal delivery (PMED). These two plasmids were also tested with co- injection of IL-4, which was anticipated to enhance B-cell response, but was found to have no effect.
  • Peptide immunizations consisted of three individual peptides, or a mixture of all three together (triple peptide- TriPep), derived from HTTexonl . All peptides were KLH conjugated to a cysteine residue added to the C terminal of each peptide.
  • Each study cohort consisted of: 1) 5 immunized R6/lhemizygous HTT transgenic mice, 2) 5 immunized zQ175 heterozygous HTT knock- in mice, 3) 5 mock treated R6/1 hemizygous HTT transgenic mice, 4) 5 mock treated zQ175 heterozygous HTT knock-in mice, and 5) 5 immunized wildtype mice.
  • Mice for both strains received an initial immunization between 6 and 8 weeks of age, and two boosts at 4-week intervals following immunization. Ten days following the last boost, the 14 to 16 week old animals were euthanized. Given these ages and the goal of determining optimal
  • PK pharmacokinetics
  • PD pharmacodynamics
  • Study groups There will be a total of 5 study groups. These are: 1) DNA plasmid N586-82Q, 2) TriPep, 3) DNA plasmid N586-82Q + TriPep, 4) DNA plasmid HTT Exonl -Q97, and 5) Untreated. In our preliminary studies we found no response and no difference among mock DNA vaccination (empty plasmid vector), mock TriPep (KLH alone in alum) and untreated (no immunization at all). Each study group will consist of 60 animals: hemizygous R6/1 females (15) and males (15), plus heterozygous zQ175 females (15) and males (15).
  • B. TriPep Immunization HTT peptides for 17, PP, and EM48 with a C- terminal cysteine will be conjugated to the hapten keyhole limpet hemocynanin (KLH) commercially.
  • KLH hapten keyhole limpet hemocynanin
  • Our initial studies optimized the dosage for prime and boost to be 100 ug.
  • Imject Alum suspension (Thermo Scientific) is used as adjuvant. Immunization is carried out in 300ul total volume (50 % alum by volume) which is delivered by sub-cutaneous injection along the back of he mouse using a different site for each injection.
  • C. DNA + TriPep Immunization Immunizations will be conducted as described for individual imunogens. Both plasmid and TriPep will be given at the same time for each prime and boost injection (TriPep once and DNA twice per immunization as described above).
  • PBMCs Peripheral mononuclear cells
  • Plasma will be collected, aliquoted and frozen for anti-HTT ELISAs.
  • Intra-cardiac perfusion with physiological saline will follow to remove blood from the brain prior to decapitation, removal and splitting of the brain into two equal hemispheres.
  • One half of the brain will be snap frozen in liquid nitrogen for later RNA and protein extraction (PARIS kit - simultaneous extraction of RNA and Protein).
  • the other brain half will be immersion fixed (4% paraformaldehyde) and archived for future analysis of HTT aggregates as informed by our results.
  • We will also archive spleen, adipose tissue, liver and pancreas for analysis in future studies beyond the scope of this application. These tissues will be divided as for brain, half fixed for histology and half frozen for future transcriptional and protein analysis.
  • Transcriptional Profile Expression analysis will be conducted to determine if active immunization has an effect of correcting HD related profile changes compared to the untreated group in brain and PBMCs. Examination of brain is contingent on detection of anti- HTT in brain lysate. Purified RNA will be subjected to array analysis using Illumina mouse gene 2.0 chips. RNA quality will be determined by use of Agilient Bioanalyzer (RIN Score > 7.0). Prohibitive cost will not allow expression analysis for all animals in all study groups. From each HD model in each immunized study group we will select the two males and females with the highest anti-HTT titers. From the untreated group we will randomly select 2 females and 2 males for each HD model for comparison.
  • ELISA For serum titers and detection of anti-HTT in brain protein isolates an indirect ELISA is performed. For capture of anti-HTT antibodies, ELISA plates are coated with 2.5 ug of HTT exonl-Q46 - MBP fusion protein (MBP-maltose binding protein). MBP is necessary for binding of the HTT protein to the plate, and also assists in presenting the HTT protein in a conformation similar to the immunogens utilized to elicit antibody production.
  • MBP-maltose binding protein HTT exonl-Q46 - MBP fusion protein
  • A. Open-field Exploratory behavior in the open field can be used to measure basic motor behavior, hyperactivity, emotionality and simple cognitive ability (Bolivar, Caldarone et al. 2000; Bolivar, Scott Ganus et al. 2002; Bolivar, Manley et al. 2003; Bolivar, Manley et al. 2004; Bolivar 2009).
  • the Smartrod Rotating Rod Apparatus (48 cm L, 1 1 cm W, 30 cm H; Accuscan Instruments, Inc., Columbus, OH) will be used to assess motor coordination. Mice are given three trials per day, with 20 min between each trial, over three consecutive days, in a modified accelerating rotorod protocol. Each trial lasts a maximum of 180 seconds. During the first 60 seconds, the rod accelerates from 0 to 15 rotations per minute (rpm). The rotation speed is kept constant at 15 rpm for the next 110 seconds, until the rod decelerates from 15 to 0 rpm in the last 10 seconds of the trial. The latency to fall is recorded for each trial and a mean latency will be calculated. The trial is terminated either when the mouse falls, as detected by photobeams at the grid floor, or at the end of the 180 sec test.
  • C. Two-Choice Swim Test In this simple visual discrimination task, the mouse must learn to associate a visual cue with a submerged platform, which allows escape from a swim tank. This task has been used to demonstrate cognition deficits in mouse models of HD, including both zQ175 and R6/2 mutants (Carter, Lione et al. 1999; Lione, Carter et al. 1999; Menalled, Kudwa et al. 2012). We will measure successful reaching of the platform, as well as escape latency and distance traveled. Each mouse will be placed in the center of a rectangular swim tank (76cm X 30.5cm X 30.5cm) filled with opaque water (6in in depth) and maintained at 25 ⁇ 1°C.
  • An escape platform 0.5cm below the surface is placed at one end of the tank, with a 60W lamp positioned over the platform, serving as a cue.
  • the opposite side of the tank is not lit for contrast.
  • the tank is surrounded by 45cm high panels made of dark grey Plexiglas, which prevents mice from using surrounding spatial cues. Mice will be given 20 trials per day (15-min inter-trial interval) until criterion is reached (18 out of 20 trials correctly reaching the platform). In each trial, the mouse will be released from the center of the tank and allowed to swim freely for a maximum of 60sec. If the mouse fails to reach the platform during that time, it is manually placed there for 5 sec. In all trials, choice as well as latency and distance traveled to reach the platform will be recorded.
  • a reversal test will be used to measure cognitive flexibility.
  • the platform will be moved to the opposite end of the tank (dark end) and mice will be given 20 trials per day (15 min inter-trial interval) until criterion is reached (18 of 20 correct platform attainments). All data will be analyzed in blocks of 5 trials. Latency and distance traveled will be measured across trials. The VideoScan 2000 tracking system (Accuscan Instruments) will be used for data acquisition. [0072]
  • mice will be re-tested once, between 24-25 weeks of age.
  • zQ175 mice will be re -tested twice, at 33-35 weeks of age and again at 50-52 weeks of age.
  • Noncytolytic, Ab-mediated clearance of viral infection has also been observed in neurons both in vivo and in vitro (Levine et al, 1991; Dietzschold et al, 1992; Byrnes et al., 2000), supporting a possible role for intracellular, Ab-mediated activity in brain.
  • Our laboratory previously demonstrated that aggregate formation of mutant N-term htt may be inhibited by binding of a sFv selected against the 17 AA residues amino-terminal to the polyQ tract (Lecerf et al, 2001), suggesting that protein binding to a region adjacent to the polyQ tract may be therapeutic. We therefore directed an immune response against the amino-terminus of htt.
  • Plasmid immunization (Robinson and Torres, 1997) was favored over peptide immunization, as polyQ-containing proteins are highly insoluble (Perutz et al, 1994). This approach allows for intracellular processing of the htt fragment and may better mimic the endogenous physiological context of htt.
  • Neurological diseases are frequently accompanied by peripheral biomarkers, abnormalities in small molecule and protein levels in tissue, serum, and urine, reflecting changes in metabolism, cell function, or protein turnover (Fain et al, 2001 ; Luthi-Carter et al, 2002; Sathasivam et al, 1999). Monitoring such indicators may facilitate diagnosis and allow routine analysis of disease progression and treatment efficacy, while eliminating the need for neuroinvasive procedures.
  • Diabetes mellitus occurs at an increased frequency in patients with HD (Farrer et al., 1985), one of a variety of unrelated genetic diseases associated with the biomarker of impaired glucose tolerance (termed syndromic diabetes, Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 2001).
  • the cause of the abnormal carbohydrate metabolism in HD remains to be fully elucidated, although recent findings from HD transgenic mice indicate that decreased insulin transcription is linked to mutant htt-mediated transcriptional dysregulation (Andreassen et al, 2002).
  • Insulin-producing pancreatic ⁇ -cells may be evolutionarily related to an insulin- producing neuron, as neurons and ⁇ -cells share common protein markers (Le Douarin, 1988; Scharfmann, 1997; Schwartz et al., 2000), and insulin-producing neurons are critical in Drosophila for normal development and metabolic control (Rulifson et al., 2002). Therefore, the neuronal dysfunction characteristic of HD may be closely related to pancreatic ⁇ -cell dysfunction.
  • Diabetes in HD patients is directly correlated with duration of HD symptoms and is manifest as hyperglycemia and hyperinsulinemia in response to glucose tolerance testing (Podolsky et al, 1972; Podolsky and Leopold, 1977).
  • a diabetic phenotype has been observed in the HD transgenic mouse model R6/2 expressing exon 1 of huntingtin (httexl) with >144 CAG repeats
  • pancreatic insulin and glucagon are dramatically decreased in the fasted state (Hurlbert et al, 1999), and pancreatic islet cells develop aggregates similar to those seen in HD brain (Sathasivam et al, 1999).
  • mice were vaccinated at 5 and 7 wks of age with 100 ug of plasmid
  • Plasmids encoded GFP pGFP; pGreen Lantern; Invitrogen
  • an HD exon 1 fragment containing the amino-terminal 17 AA plus 25Q or 103Q fused to GFP pHD25Q- GFP, nonpathogenic, normal-length polyQ; pHD103Q-GFP, pathogenic, expanded polyQ
  • CMV cytomegalovirus
  • MATLEKLMKAFESLKSFC (SEQ ID NO:3) (Wadsworth Center Peptide Core facility) conjugated to cBSA (according to manufacturer's protocol, Pierce) in 100 uL 25% Alum adjuvant (Pierce) in PBS from 5 to 9 wks of age.
  • DRPLA62Q-GST (donated by Intraimmune Therapies), or HD103Q-GST by calcium phosphate precipitation (general methods).
  • Cells were lysed in RIPA buffer, and lysate was used for SDS-PAGE, 10% gel (general methods).
  • Nitrocellulose membrane was blocked using 10% nonfat dry milk in 0.1% Tween/PBS for 1 h at room temp., and serum (1 : 100) was diluted in blocking solution to probe for the plasmid-encoded proteins.
  • Anti-GFP (1 1E5; 1 : 1000; Quantum) and anti-GST (B-14; 1 : 1000; Santa Cruz) mouse Abs were used as positive controls.
  • mice were tested at 12 wks of age. Food was removed ⁇ 5 PM, and overnight fasting blood glucose was measured 16-18 h later. Mice were then given an intraperitoneal bolus injection of glucose in PBS at 2 mg glucose/g body weight (as per clinical protocol). Glucose clearance was initially measured by the traditional clinical method of taking periodic readings (30 min., 1 h, 2 h, and 6 h postinjection). We subsequently determined that readings at 2 h were sufficient to demonstrate differences in the ability to deal with glucose load. Mice that weighed ⁇ 14 g or had an average blood glucose ⁇ 48 mg/dl (based on the lowest wild- type reading obtained) were not included in the analysis.
  • mice were included in only the daytime fasting blood glucose assay or the glucose tolerance assay. Only female mice were used in the analysis. Comparisons between groups were made using the Mann-Whitney U test. P levels ⁇ 0.05 were considered significant.
  • mice in the fed and 6-h-fasted states were transcardially perfused with 4%
  • PFA/PBS at 12.5-13 wks of age in the late afternoon.
  • Pancreas was harvested, paraffin- embedded, and sectioned at 5 um thickness. Sections were deparaffinized in xylene and rehydrated through a graded ethanol series (general methods). Insulin staining was performed using rabbit-anti-insulin Ab (1 :500-1 : 1000; Santa Cruz) followed by Alexa 488-donkey-anti- rabbit Ab (1 :800-1 : 1000; Molecular Probes).
  • htt immunostaining slices were probed with 3B5H10 mouse-anti-polyQ Ab (1 : 100-1 :500) or S830 sheep-anti-httexl Ab (1 : 1,000; a gift of G.
  • mice bilaterally plasmid-injected mice, using saline vehicle, are described below.
  • GFP were classified as "responsive" to vaccination overall, with the understanding that Ab against some critical epitopes may not be discernible by this Western blot screening assay. All immunoresponsive mice possessed Ab against all three screening proteins (GFP, HD25Q- GFP, HD103Q-GFP). We observed htt- and polyQ-directed Abs, capable of binding
  • mice without detectable anti-htt antibodies sometimes exhibited diabetic protection (discussed below, Fig. 4).
  • Preliminary vaccinations with a pHD25Q-GFP plasmid had elicited a much lower rate of detectable Ab response; therefore, all subsequent HD plasmid vaccinations were performed using pHD103Q-GFP.
  • pHD103Q-GFP-immunoresponsive R6/2 mice clearly had better control of fasting blood glucose than did pHD103Q-GFP-nonresponsive or pGFP-treated R6/2 mice (both P ⁇ 0.01). Examined on an individual basis, untreated R6/2 mice showed a clear progression toward fasting hyperglycemia.
  • Glucose tolerance is a more rigorous test for diabetes than is 6-h fasting blood glucose because it challenges the body, forcing it to handle a large glucose load following overnight fasting (16-18 h), a procedure that should normalize blood glucose levels more effectively than does daytime fasting, particularly since mice are most active at night.
  • pHD103Q-GFP immunoresponsive R6/2 mice had better blood glucose control than did other R6/2 treatment groups.
  • wild-type and pHD103Q-GFP-responsive R6/2 mice were not significantly different, while all other R6/2 treatment groups were statistically different from the wild-type group (Fig. 4C; all P ⁇ 0.02).
  • pHD103Q-GFP-immunoresponsive R6/2 mice maintained a near-normal ability to clear glucose from the bloodstream, while pHD103Q-GFP-nonresponsive and control-treated R6/2 mice remained glucose intolerant.
  • R6/2 diabetic phenotype (data not shown).
  • R6/2 mice were immunized weekly with 3B5H10 Ab and glucose tolerance tested at 12 wks of age. This treatment showed no benefit, as overnight fasting blood glucose levels were near the wild-type range (120-200 mg/dL), but 2 h blood glucose levels were more characteristic of untreated R6/2 mice (>200 mg/dL).
  • pGFP-treated R6/2 mice also had significantly lower blood glucose than untreated R6/2 mice at both the 0- and 2-h time points (Fig. 4B and Fig. 4C; P ⁇ 0.01).
  • CpG immunization had no effect on wild-type mice. This suggests that the protective effects seen with control pGFP plasmid vaccination, and some of the effects observed in pHD103Q-GFP-immunoresponsive mice, may be due to nonspecific, CpG-directed stimulation of the innate immune system.
  • the R6/2 Diabetic Phenotype May Be Caused by Insulin Deficiency, Largely Corrected with Response to HD Plasmid Vaccination
  • pancreatic tissue histologically to determine a relationship between pancreatic insulin staining, diabetic phenotype, and immunoresponsiveness to HD plasmid vaccination In mice perfused in the fed state 2-4 d after blood glucose testing, I noted an imperfect correlation between 6-h fasting blood glucose levels and insulin immunoreactivity. Although wild-type mice showed strong staining throughout islets, there was variability between mice. I tried starving wild-type mice for 6 h prior to perfusion to help normalize blood glucose, and possibly pancreatic insulin levels, but this did not increase the correlation between blood glucose and insulin immunoreactivity. In immunostaining of pancreas harvested from mice in the fed state, performed by W.J.
  • immunoresponsive mice may possess Abs that recognize denatured httexl by Western blotting but not native httexl in tissue, or that anti-htt Ab levels were insufficient for detection by this method.
  • Fain and coworkers did not detect diabetes in their R6/2 mice, although they did not normalize blood glucose by fasting, challenge with glucose tolerance testing, or measure blood glucose in mice >10 wks of age. In addition to assay variations, it is possible that genetic differences between R6/2 colonies may contribute to the inconsistencies between our findings and those of others. Sizing of the CAG repeat (Wadsworth Center Molecular Genetics core) in our R6/2 mice revealed >170 repeats, an expansion beyond the 144 repeats reported in the original colony (Mangiarini et al., 1996) due to instability at the transgene locus.
  • the R6/2 diabetic phenotype appears to be caused by an insulin defect in pancreatic ⁇ -cells, as determined by immunohistochemical analysis. Recent evidence provides a link between decreased insulin transcription and mutant htt-mediated
  • R6/2 islets were consistently different from wild-type, exhibiting overall decreased insulin staining with heterogeneity due to strong staining in a minority of cells. We much less frequently noted a staining pattern similar to that of Hurlbert and colleagues (1999), who observed that R6/2 islets had decreased insulin staining of a homogeneous nature. pHD103Q-GFP-immunoresponsive R6/2 mice had clearly enhanced insulin staining compared to nonresponsive or control-treated subjects, in agreement with improved glucose tolerance among immunoresponsive R6/2 mice. The few strongly insulin-positive cells of untreated and control-treated R6/2 mice may be capable of only weak blood glucose regulation. The differences that we observed between 6-h and overnight fasting may reflect such incomplete blood glucose control, suggesting that, with sufficient time in the absence of glucose influx from eating, these diabetic, glucose-intolerant mice might produce sufficient insulin to normalize their blood glucose levels.
  • the htt-specific diabetic correction may be a function of both the presentation context and the antigenicity of the htt protein fragment.
  • Plasmid vaccination allows intracellular processing of the encoded protein and MHC class I presentation, which should provide immune presentation of the htt fragment in a conformation similar to that of native htt, as well as a strong TH1 bias (Krieg and Davis, 2001).
  • the HD17AA peptide vaccination utilizes the MHC class II pathway and thus a more TH2-type response. This difference may evoke divergent Ab repertoires of epitope recognition.
  • One relevant immunogenic epitope might be the expanded polyQ tract of HD103Q-GFP.
  • mice that were classified as "responsive" to vaccination may not have been producing Ab against the critical epitope in HD103Q-GFP.
  • Our Western blot screening approach was not designed for epitope determination, but instead to detect a generalized immune response to the plasmid-encoded protein.
  • Our data did not resolve the critical epitope of HD103Q-GFP, as htt- and polyQ-specific antibodies were not observed in all nondiabetic R6/2 mice classified as immunoresponsive (with Abs against GFP, HD25Q- GFP, and HD103Q-GFP).
  • the epitope availability in vivo differs from that on a Western blot.
  • HD tissue Screening against other forms of HD tissue may help to elucidate the significant epitope of HD103Q-GFP; however, probing R6/2 pancreatic sections with sera from immunoresponsive mice did not detect htt-positive islets. Equally important may be the role of T-cells activated by MHC class I presentation. Although overt cytolytic or inflammatory processes were not observed, the cytokine milieu in the pancreas attributable to the T-cell arm elicited by DNA vaccination might serve to stimulate insulin production, improve insulin regulation, or mitigate inhibitory effects of mutant htt. It is also possible that cytokines may upregulate ⁇ -cell turnover, with overall improved function of the islet population.
  • the pGFP plasmid- and CpG oligonucleotide-treated R6/2 mice were less completely corrected than were the pHD103Q-GFP-immunoresponsive R6/2 mice. It is possible that bacterial endotoxins contained in the plasmid preparations could also stimulate an immune response; however, the CpG oligonucleotides, which elicited effects similar to those of pGFP plasmid, were synthesized in vitro.
  • BBB blood-brain barrier
  • Brain capillary endothelial tight junctions provide the central nervous system with an immune- privileged environment (Rabchevsky et al, 1999).
  • a low level of Ab is normally present in the cerebrospinal fluid, which remains unchanged upon bodily response to peripheral hyperimmunization (Cserr et al, 1992).
  • peripheral passive immunization with anti-amyloid- ⁇ Ab led to brain amyloid plaque decoration with Ab and subsequent clearance (Bard et al, 2000).
  • Immunization against neurotoxic proteins may provide an avenue for treatment of a variety of disorders utilizing the host immune system.
  • Immunization against Alzheimer's disease using amyloid- ⁇ peptides has shown great promise with brain amyloid plaque clearance and memory recovery in animal models (Schenk et al., 1999; Morgan et al, 2000; Janus et al., 2000; Dodart et al., 2002), although results from clinical trials suggest that active immunization may elicit deleterious side effects (Birmingham and Frantz, 2002).
  • prion protein is an endogenous species.
  • Alzheimerdisease-like pathology in the PDAPP mouse Nature 400: 173-177.
  • a beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease. Nature 408: 979-982.
  • Immunotherapy both active and passive, is increasingly recognized as a powerful approach to a wide range of diseases, including Alzheimer's and Parkinson's.
  • Huntington's disease an autosomal donant disorder triggered by misfolding of huntingtin (HTT) protein with an expanded polyglutamine tract, could also benefit from this approach.
  • Individuals can be identified genetically at the earliest stages of disease, and there may be particular benefits to a therapy that can target peripheral tissues in addition to brain.
  • active vaccination study we first examined safety and immunogenicity for a broad series of peptide, protein, and DNA plasmid immunization protocols, using fragment (R6/1), and knock-in (zQ175) models. No safety issues were found. The strongest and most uniform immune response was to a combination of three non-overlapping H7Y Exonl coded peptides, conjugated to KLH, delivered with alum adjuvant.
  • N586-82Q plasmid delivered via gene gun, also showed ELISA responses, mainly in the zQ175 strain, but with more variability, and less robust responses in HD compared to wild-type controls.
  • Transcriptome profiling of spleens from the triple peptide-immunized cohort showed substantial HD-specific differences including differential activation of genes associated with innate immune responses, absence of negative feedback control of gene expression by regulators, a temporal dysregulation of innate immune responses, and transcriptional repression of genes associated with memory T cell responses.
  • the immune system is increasingly recognized as a player in the pathogenic cascade of neurodegenerative diseases triggered by misfolding proteins, as well as a potential source of valuable targeted immunotherapies.
  • Applying immunotherapeutic approaches to Alzheimer's and Parkinson's disease has shown significant promise in animal models, although most reported human trials to date have not reached efficacy criteria, possibly due to a combination of a lack of early enough biomarkers to identify cases, and heterogeneity of the underlying disease mechanisms within the broad neurological diagnoses (I, 2).
  • Huntington's disease (HD) is an autosomal dominant disorder that circumvents most of the above problems, since genetic diagnosis can be done at a relatively early pre-manifest stage, and the underlying primary gene defect is more uniform than for the neurodegenerations tested to date.
  • the HD mutation is a trinucleotide repeat expansion leading to an increase in the length of a polyglutamine (polyQ) tract at the N terminus of the huntingtin (HTT) protein (3).
  • polyQ polyglutamine
  • HTT huntingtin
  • 49PQLPQPPPQAQP60 [used by the Patterson lab to select Happl and Happ3 (21)]; and 74PPPGPAVAEEPLHRP88, the putative EM48 epitope. All peptides were synthesized and KLH conjugated commercially (GenScript, Piscataway, NJ). The latter is human-specific, and Wang et al, 2008, showed suppression of neuropil aggregates and neurological symptoms when a scFv intracellular antibody form of this monoclonal was very highly expressed from an adenovirus in striatum (22). Mice were immunized sub-cutaneously (sc) with 200 ul total volume (lOOug of each peptide).
  • HTT Exonl-46Q protein linked to maltose binding protein was provided by CHDI, and cleaved and MBP removed just prior to immunizations with a Thrombin CleanCleaveTM Kit (Sigma, St. Louis, MO). Sc injections of 50ug of protein in Alum were done within one hour of completing the cleavage/clean-up reaction.
  • H7T Exonl-97Q carries the full H7T Exonl and has been used extensively, while N586-82Q covers significant additional HTT sequence, and has been used in mice and in cell cultures (50).
  • Delivery used a particle mediated epidermal delivery (PMED) device, developed by Dr. Deborah Fuller, for the efficient epidermal delivery of the plasmid DNA (51).
  • the Fuller lab (Univ. of Washington) generously produced the delivery cartridges from plasmids supplied by the Messer lab.
  • Hrr Exonl -97Q or N586-82Q were delivered for each immunization (2ug at each at separate sites in the shaved abdomen).
  • 114 was co-administered (0.4ug) having been mixed with either of the two plasmids prior to PMED cartridge preparation.
  • This protocol was tested due to success in a range of vaccine studies (52), It combines the presentation of a presumably wider range of epitopes elicited when the protein is synthesized intracellularly with the stronger antibody responses that characterize protein/peptide immunogens. Plasmid was used for primary and the first boost, and protein (or peptide) for the second boost. Immunizations were conducted as described above for either sc. or PMED delivery.
  • Anti-huntingtin antibodies in sera of immunized mice were measured by ELISA (53). Briefly, ELISA Maxisorp (Nunc) 96-well plates were coated with 25 ug/ml of MBP-Exon 1 in PBS and incubated at 4°C overnight. Plates were washed three times with PBS, 0.25% BSA, and 0.05% Tween 20 and blocked with PBS, 0.25% BSA, 0.05% Tween, and 1 mM EDTA for 1 hour at room temperature. Serial-fold dilutions of sera in blocking buffer were added to the wells. Samples added to wells without bound antigen served as negative controls.
  • ST14A cells were transiently transfected with a plasmid expressing Exon 1 of
  • Electrophoretic transfer of proteins to a PVDF membrane was accomplished using the semi- dry transfer method and a buffer consisting of 25 mM Tris-HCl, 192 mM glycine, 20% methanol.
  • the membrane was subsequently blocked in 5% non-fat dry milk in Tris buffered saline (TBS)-Tween 20 for one hour at room temperature and incubated with appropriate dilutions of serum overnight at 4°C.
  • Bound anti-HTT antibody was detected after a 1 hour incubation with horseradish peroxidase conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) followed by chemiluminescent detection.
  • Spleens were dissociated by mechanical disruption and red blood cells were removed by hypotonic lysis in 0.15 M ammonium chloride.
  • Splenocytes were lysed in RLT buffer (Qiagen, Valencia, CA) and cell lysates were homogenized using a QIAshredder homogenizer (Qiagen).
  • Cellular RNA was purified using RNeasy columns (Qiagen) and residual genomic DNA was digested with RQ1 DNase (Promega, Madison, WI). RNA was precipitated and re-suspended in sterile water. RNA integrity was verified by agarose gel electrophoresis. RNA quality and concentration were measured using an Agilent 2100 Bioanalyzer (Life Technologies).
  • cRNA was prepared according to the Affymetrix standard protocol as described (54) and hybridized to Affymetrix Mouse Gene 2.0 ST by SU YMAC (Syracuse, NY.)
  • the 46Q protein had a strong tendency to begin aggregating almost immediately upon cleavage from the MBP, which had the advantage of testing a different set of available epitopes, but also made the solution very non-homogeneous, and challenging to work with.
  • Table 1 A combination of three non-overlapping HTT exonl peptides is highly immunogenic in HD mutant and control mice.
  • Anti-Htt antibodies in sera of immunized mice were measured by ELISA or Western blotting.
  • Antibody titers > 1000 are used to indicate relative responses, and are shaded in grey.
  • Serum antibody titer is defined as the reciptorcal of the dilution whose absorbence is 3X that of negative controls.
  • HTT N586-Q82 plasmid DNA induced lower antibody titers in HD mutant mice than in WT controls (Fig. 6).
  • H7YN586-Q82 was more immunogenic than HTT Exonl -Q97 (Table 2).
  • Two strategies were used to augment the immunogenicity of the HTT plasmid DNAs.
  • a cytokine adjuvant an 114 plasmid DNA, mouse origin (23) was co-administered with the HTT plasmid DNA.
  • the 114 plasmid did not have an adjuvant effect in WT mice immunized with either of the two HTT plasmids, or in the HD mouse model after immunization with HTT exonl -Q97, Surprisingly, co-administration of the 114 plasmid had an inhibitory effect on antibody titers in the 2 HD mouse models after immunization with the H7YN586-Q82 plasmid.
  • the ⁇ N586-Q82 plasmid is more immunogenic than the ⁇ exonl -Q97 plasmid in HD mutant and control mice.
  • HTT N586-Q82 plasmid is more immunogenic than the HTT exonl-Q97 plasmid in HD mutant and control mice.
  • Anti-Htt antibodies in sera of immunized mice were measured by ELISA or Western blotting, as in Table 1.
  • the other approach to increase the immunogenicity ⁇ plasmid DNA relied on including a non-plasmid immunogen as a vaccine boost.
  • the vaccine boost chosen for this portion of the work was the combination of three non-overlapping HTT exon 1 peptides which is the most immunogenic of all regimens tested in this series (Fig. 1).
  • the heterologous vaccines consisting of a priming dose with the HTT plasmid DNA followed by two boosts with the triple peptides did not augment antibody responses (Table 2) suggesting that different epitopes are being recognized in the priming and boosting immunogens.
  • Reactome, Immunologic signatures, and GeneOntology were analyzed to identify those that correlated with strain differences in unimmunized mice or immunization differences in mutant HD and WT mice.
  • 11 correlated with strain differences in unimmunized mice, none of which is involved in innate immunity (manuscript in preparation), while 15 correlated with immunization differences in mutant and WT mice (Table 3).
  • 15 correlated with immunization differences in mutant and WT mice (Table 3).
  • Supplemental Table 1 Of the 15 gene sets, 1 1 showed positive enrichment scores (Supplemental Table 2), and four showed negative enrichment scores (Supplemental Table 3).
  • An example of a GSEA result Biocarta IL22BP is shown in Figs. 7A-7B.
  • Table 3 Fifteen gene sets in the MSigDB (Kegg, Reactome, Immunologic signatures, and Gene Ontology) correlated with immunization differences in HD mutant and wild-type mice. Gene sets that correlate with immunization differences in mutant HD mice function in proinflammatory pathways
  • the 15 gene sets include biologically informative sets for nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activation, antigen processing, cytokine signaling, and macrophage activation (Table 5).
  • NFkB nuclear factor kappa-light-chain-enhancer of activated B cells
  • Table 5 macrophage activation
  • NFkB activation, cytokine signaling, and pro-inflammatory macrophage responses were positively correlated with immunized mutant mice while antigen processing and anti-inflammatory macrophage responses were negatively correlated with immunized mutant mice.
  • Immunized zQ175 mice showed up-regulation of NFkB and proinflammatory cytokine gene sets while immunized R61 mice showed up-regulation of pro-inflammatory macrophage gene sets when compared to immunized wild-type mice.
  • Up-regulation of proinflammatory gene sets in immunized mutant HD mice was accompanied by down- regulation of anti-inflammatory macrophage gene sets when compared to immunized wild- type mice (Table 6).
  • Table 4 Gene sets that correlate with immunization differences in mutant HD mice function in proinflammatory pathways. The fifteen gene sets (in Table 3) were organized into biologically functional groupings. Four functional groupings were identified. Table 5. Individual immune-related genes are differentially expressed after immunization of mutant HD and wild-type mice
  • Table 5 Individual immune-related genes are differentially expressed after immunization of mutant HD and wild-type mice. Genes in the leading-edge subset from eight gene sets (in table 3) for immunized mutant versus immunized wild-type omparisons were sorted based on function and assigned to two categories, innate immune responses or T/B cell responses.
  • inflammatory responses to LPS TNFa signaling via NFKB targets
  • NOD-like receptor signaling which drives activation of NFKB and MAPK
  • dendritic cell maturation in response to inflammatory stimuli Two gene sets highlight additional immune responses; IL23 signaling which is involved in development of Thl7 responses and diverse immune responses in Reactome immune system.
  • the leading-edge subset can be interpreted as the core of a gene set that accounts for the enrichment signal (24). Examination of genes in the leading-edge subset often reveals additional biologically informative sets. We undertook an analysis of immune- related genes in the leading edge subsets from gene sets that correlated with immunization of mutant mice to identify additional biologically informative sets.
  • the leading edge subsets from eight gene sets for immunized mutant versus immunized wild-type comparisons were collapsed into four groupings; Imm R61>Imm WT, Imm zQ175>Imm WT, Imm R61 ⁇ Imm WT, and Imm zQ175 ⁇ Imm WT.
  • mice were sorted based on function and assigned to pro-inflammatory responses, anti-inflammatory responses, or T/B cell responses (Table 5).
  • immunized zQ175 mice showed up-regulation of genes that function in both pro-inflammatory and anti-inflammatory responses (Table 5).
  • Genes associated with pro-inflammatory responses included those encoding cytokines, chemokines, related receptors, and a Toll-like receptor.
  • Genes associated with anti-inflammatory responses encoded negative regulators of inflammation.
  • Immunized mutant mice also exhibited down- regulation of genes that function in Th2 responses and memory T cell responses.
  • the CBAB6F1 genetic background was chosen to allow responses driven by two diverse H-2 haplotypes, while preserving uniform genetic backgrounds.
  • the study as a whole did reveal classes of more uniform non-responsive immunization protocols that can be ruled out. It is clear that plasmid 114, which has been used as an adjuvant in infectious disease plasmid immunization studies (23), does not improve responses in these trials.
  • the single-chain variable region antibody, C4scFv delivered as a gene, can modulate phenotypic effects of the mutant HTT Exonl fragment in cells, drosophila, and mouse striatum.
  • the effects are short-term, and over time the misfolded protein accumulates, even in the continuous presence of an antibody fragment that binds to HTT AA1-17 (25).
  • Kinetically, the mutant protein can misfold during brief windows of antibody dissociation, and will eventually form aggregates that cannot reversibly re-fold.
  • the mutant HD gene is expressed ubiquitously, and the necessity to counteract long-term accumulation may be less critical in the peripheral and non-neuronal cells that can be targeted with an active vaccination strategy.
  • Active vaccination was able to modulate the peripheral pancreatic insufficiency phenotype in the severe R6/2 mouse model, with plasmid correcting more completely than AA1-17 peptide, despite a lower level of antibody as measured by WB (11).
  • immunogenicity of peptides is affected by differences in a variety of cellular processes including uptake of peptides, processing and presentation of peptides by major histocompatibility complex (MHC) class II molecules in antigen presenting cells to T helper cells.
  • MHC major histocompatibility complex
  • uptake of plasmid immunogens results in intracellular expression of peptides which traffic through the MHC class I pathway.
  • these processes may differ in the two mutant mouse strains.
  • defective internal cellular transport processes in T cells in the two mutant mouse strains may also affect the immunogenicity of plasmid immunogens because of data that dynein-mediated axonal transport is defective in HD (27), and similar proteins appear critical for the T cell synapse (28).
  • NFKB protein kinase B
  • Illb, Tnf, Nfkbia, and Tnfaip3 are target genes for the transcription factor, NFKB I, with IL IB and TNF serving as positive regulators of gene expression while NFKBIA and TNFAIP3 are negative regulators of gene expression (KEGG database; (34, 35) ⁇
  • target genes for TNF include Illb, 116 and Tnf (positive regulators) along with NFKBIA and TNFAIP3 (negative regulators) (34, 35).
  • genes encoding both positive and negative regulators in the five signaling pathways are detected simultaneously, implying a failure in negative feedback mechanisms to inhibit expression of pro-inflammatory genes.
  • the sustained expression of genes associated with innate immunity ten weeks after the initial immunization, suggests a failure in the temporal control of innate immune responses. These responses were not unexpected, although the comparison of the two mutant strains is informative.
  • Aifl is expressed in macrophages and is a key regulator of inflammation, influencing cytokine production, inflammatory mediators, and expression of adhesion molecules (36).
  • AIF 1 is also present in activated T cells and up-regulates IL4 production.
  • Both Aifl and Il4r are down- modulated in immunized zQ175 versus immunized wild-type mice.
  • NFATc2 is a member of the NFAT family of transcription factors. NFAT signaling modulates the function of cells involved in innate immune responses such as macrophages, dendritic cells, mast cells, neutrophils, and natural killer cells (37). In addition, NFAT influences lymphocyte development, activation, and T cell differentiation and is expressed at higher levels in memory T cells (38). FCGR1 is expressed on macrophages and binds IgG with high affinity resulting in activation of phagocytic activity (39). Two additional genes, Fyb and Lairl, are down-regulated in immunized R6/1 versus immunized wild-type mice.
  • the plasmid immunogens differ, one hypothesis would be that the previous plasmid induced a more "protective" antibody response. It will be particularly interesting to determine whether the high antibody titers achieved with the new triple peptide protocol provide functional protection. This can then be compared to protection from the lower, but still measurable, titers produced with plasmid vaccination. Individual peptides can also be tested, although our preliminary data suggested that immunizing with a combination of the three peptides gave a much more robust response that single peptides alone.
  • Huntington's Disease a genome -wide perspective. Mol Neurobiol, 51, 406-423. Seredenina, T. and Luthi-Carter, R. (2012) What have we learned from gene expression profiles in Huntington's disease? Neurobiol Dis, 45, 83-98.
  • Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease. JNeurosci, 29, 13589-13602.
  • Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines. J Virol, 76, 4536-4546.
  • Immature hematopoietic cells display selective requirements for adhesion- and degranulation-promoting adaptor protein in development and homeostatsis. Eur J Immunol, 37, 3208-3219.
  • IL-10 is pathogenic during the development of coxsackievirus B4-induced chronic pancreatitis. Virology 395(1): 77-86. 19800092

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Abstract

L'invention concerne, entre autres, des agents thérapeutiques et des procédés thérapeutiques permettant de traiter la maladie de Huntington (HD).<i /> Dans un aspect, l'invention concerne l'utilisation de plasmides dérivés de l'Huntingtine et de peptides et pour une immunisation active, en tant qu'agent thérapeutique de la maladie de Huntington (HD). Dans un mode de réalisation, l'immunogène utilisé comme agent thérapeutique comprend un peptide de protéine HTT humaine. Dans des modes de réalisation particuliers, le peptide de protéine HTT humaine est choisi dans le groupe constitué de N17 (17 premiers résidus d'exon1), PP (résidus 49 à 60), et EM48 (résidus 74 à 88). Dans d'autres modes de réalisation, l'immunogène comprend une combinaison de peptides de la protéine HTT humaine, comprenant, sans limitation, la combinaison de peptides de la protéine HTT humaine comprenant au moins l'un des peptides choisis dans le groupe constitué de N17, PP, et EM48.
PCT/US2015/049025 2014-09-05 2015-09-08 Utilisation de plasmides et peptides dérivés de l'huntingtine pour une immunisation active comme agent thérapeutique de la maladie de huntington (hd) WO2016037191A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9605019B2 (en) 2011-07-19 2017-03-28 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
US9617547B2 (en) 2012-07-13 2017-04-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant
US9695211B2 (en) 2008-12-02 2017-07-04 Wave Life Sciences Japan, Inc. Method for the synthesis of phosphorus atom modified nucleic acids
US9982257B2 (en) 2012-07-13 2018-05-29 Wave Life Sciences Ltd. Chiral control
US10144933B2 (en) 2014-01-15 2018-12-04 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator
US10149905B2 (en) 2014-01-15 2018-12-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having antitumor effect and antitumor agent
US10160969B2 (en) 2014-01-16 2018-12-25 Wave Life Sciences Ltd. Chiral design
US10167309B2 (en) 2012-07-13 2019-01-01 Wave Life Sciences Ltd. Asymmetric auxiliary group
US10307434B2 (en) 2009-07-06 2019-06-04 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
US10322173B2 (en) 2014-01-15 2019-06-18 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent
US10428019B2 (en) 2010-09-24 2019-10-01 Wave Life Sciences Ltd. Chiral auxiliaries

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BUTLER, D ET AL.: "Engineered Antibody Therapies To Counteract Mutant Huntingtin And Related Toxic Intracellular Proteins. Progress In Neurobiology", THE NEUROTOXICITY OF MUTANT PROTEINS);, vol. 97, 1 May 2012 (2012-05-01), pages 190 - 204, [retrieved on 20151220] *
GRAHAM, R ET AL.: "Cleavage At The 586 Amino Acid Caspase-6 Site In Mutant Huntingtin Influences Caspase-6 Activation In Vivo.", THE JOUMAL OF NEUROSCIENCE: THE OFFICIAL JOURNAL OF THE SOCIETY FOR NEUROSCIENCE, vol. 30, no. 45, 10 November 2010 (2010-11-10), pages 15019 - 15029, [retrieved on 20151220] *
GRAY, M ET AL.: "Full-Length Human Mutant Huntingtin With A Stable Polyglutamine Repeat Can Elicit Progressive And Selective Neuropathogenesis In BACHD Mice.", THE JOUMAL OF NEUROSCIENCE: THE OFFICIAL JOURNAL OF THE SOCIETY FOR NEUROSCIENCE, vol. 28, no. 24, 11 June 2008 (2008-06-11), pages 6182 - 6195, [retrieved on 20151220] *
MAY R ET AL.: "Antibodies To Keyhole Limpet Hemocyanin Cross-React With An Epitope On The Polysaccharide Capsule Of Cryptococcus Neoformans And Other Carbohydrates: Implications For Vaccine Development.", JOURNAL OF IMMUNOLOGY, vol. 171, no. 9, 1 November 2003 (2003-11-01), Baltimore, MD, pages 4905 - 4912, [retrieved on 20151220] *
MESSER A ET AL.: "Intrabodies As Neuroprotective Therapeutics.", NEUROTHERAPEUTICS, vol. 10, 7 May 2013 (2013-05-07), pages 447 - 458, [retrieved on 20151220] *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9695211B2 (en) 2008-12-02 2017-07-04 Wave Life Sciences Japan, Inc. Method for the synthesis of phosphorus atom modified nucleic acids
US10329318B2 (en) 2008-12-02 2019-06-25 Wave Life Sciences Ltd. Method for the synthesis of phosphorus atom modified nucleic acids
US10307434B2 (en) 2009-07-06 2019-06-04 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
US10428019B2 (en) 2010-09-24 2019-10-01 Wave Life Sciences Ltd. Chiral auxiliaries
US10280192B2 (en) 2011-07-19 2019-05-07 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
US9605019B2 (en) 2011-07-19 2017-03-28 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
US9982257B2 (en) 2012-07-13 2018-05-29 Wave Life Sciences Ltd. Chiral control
US10167309B2 (en) 2012-07-13 2019-01-01 Wave Life Sciences Ltd. Asymmetric auxiliary group
US9617547B2 (en) 2012-07-13 2017-04-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant
US10590413B2 (en) 2012-07-13 2020-03-17 Wave Life Sciences Ltd. Chiral control
US10149905B2 (en) 2014-01-15 2018-12-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having antitumor effect and antitumor agent
US10322173B2 (en) 2014-01-15 2019-06-18 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent
US10144933B2 (en) 2014-01-15 2018-12-04 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator
US10160969B2 (en) 2014-01-16 2018-12-25 Wave Life Sciences Ltd. Chiral design

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