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Definitions

• Adeno-associated viral (AAV) vectors are often used in gene therapy for neurological disorders because of its safety profile and promising results in clinical trials.
• AAV-based gene therapy is effective transduction of large numbers of the appropriate cell type.
• engineered AAV vectors of any preceding aspect further comprising a first expression cassette comprising a regulatory element (such as, for example, a woodchuck posttranscriptional regulatory element (WPRE) sequence) and a transgene (such as, for example, ⁇ -Galactosidase 1 (GLB1), Niemann-Pick C1 (NPC1), Apolipoprotein E (APOE), GD3 synthase, huntingtin (Htt), interleukin (IL)-10 (IL-10), Myelin Oligodendrocyte Glycoprotein (MOG), mitogen-activated protein kinase 8 interacting protein 3 (MAPKA8IP3), survival motor neuron (SMN) 1 (SMN1), SMN2, Cas9, ⁇ -Glucocerebrosidase (GBA), Sphingomyelin phosphodiesterase 1 (SMPD1), beta-hexosaminidase A (HEXA), nerve growth factor (NGF
• a regulatory element
• the AAV vector has at least a 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75% efficiency of transduction. Also disclosed are methods of delivering a gene to neural tissue wherein the AAV vector is administered systemically (such as, for example, intravenously, including but not limited to, i.v. injection or i.v. drip; and/or retro-orbitally); or via cerebrospinal fluid injection. 7.
• Figure 11 shows representative image for neuronal tropism assessment (NeuN). Double immunofluorescence staining against GFP (transgene, green) and the specific neuronal marker NeuN (red). White arrow heads indicate NeuN/GFP colocalization confirming the neuronal tropism of LC.V1 vector.
• Rec2 capsid One concern of the Rec2 capsid is its high efficiency for liver transduction that hampers needs of selective gene transfer of the adipose tissue.
• Rec2 In order to generate new serotypes with improved adipose-tropism and eliminating liver transduction, several point mutations were made in the capsid of Rec2 by analyzing amino acid sequence of capsid among AAV8, Rec2, Rec3, and AAV2. 48.
• Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
• D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such.
• Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type e.g., D-lysine in place of L- lysine
• D-amino acid of the same type e.g., D-lysine in place of L- lysine
• the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
• the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol.
• the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). a) Pharmaceutically Acceptable Carriers 91.
• the compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier. 92.
• Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
• Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art. 94.
• Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
• the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are affected.
• the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
• the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
• the dosage can be adjusted by the individual physician in the event of any counterindications.
• the therapeutic agent can also comprise a microRNA including but not limited to miRNA-222, miRNA-7, and miRNA-132.
• a microRNA including but not limited to miRNA-222, miRNA-7, and miRNA-132.
• the disclosed AAV vectors can encode any peptide, protein, antibody, or nucleic acid appropriate for the treatment of the neurological disease or condition. Examples of such nucleic acids, peptides, proteins, antibodies known to those of skill in the art. D.
• Example 104 The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in qC or is at ambient temperature, and pressure is at or near atmospheric. 1.
• Example 1 Generation of LC.V1 capsid 105.
• AAV vectors have become an attractive gene delivery vehicle because they can transduce both dividing and post-mitotic tissues with low immunogenicity and long-lasting transgene expression.
• AAV2-mediated gene transfer to tissues such as liver, muscle, retina and central nervous system has been reported.
• new non-human serotypes such as AAV8 were isolated and identified through PCR-based screening of primate tissues, and was shown to transduce neurons better than that of AAV2.
• comparison among these new primate serotypes revealed widespread neuronal transduction following infusion of cy5, rh20 and rh39, to a level greater than that of AAV8.
• Rec2 capsid a series of hybrid recombinant capsids, including Rec2 capsid, were generated by structural domain exchange or shuffling among fragments from cy5, rh20 and rh39, in hope that their tropisms could render the novel hybrid serotypes efficiently target retina.
• In vivo and in vitro evaluations showed the transduction efficacy of Rec2 capsid no better than that of AAV2 or AAV5.
• Rec2 serotype exhibits widespread transduction in both brown and white adipose tissue superior to the naturally occurring serotypes tested including AAV1, AAV8, and AAV9 serotypes.
• Rec2 serotype vectors have been applied in basic and translation research.
• Rec2 can also transduce liver effectively via intravenous injection.
• Capsid (cap) gene in AAV2 genome via an alternative splicing and initiation encodes structural viral proteins (VPs), including VP1, VP2 and VP3, thus all three VP proteins share identical carboxyl-terminal amino acids.
• the variant 1, namely LC.V1 capsid displayed high transduction to neurons and widespread transgene expression in mouse brain (Fig 1) and rat brain (Fig 2, 3).
• LC.V1 vector expressing green fluorescent protein (GFP) was injected to the striatum of C57BL/6 mouse unilaterally at the dose of 1 x 10 9 viral particles in 1 PL.
• the transduction range was ⁇ 3 mm which is approximately 15% of the mouse brain (Fig 1).
• LC.V1 vector displayed similar widespread transduction in Sprague- Dawley rat brain via Convection-enhanced Delivery (CED) to striatum unilaterally (1.8 x 10 11 viral particles in 15 PL).
• CED Convection-enhanced Delivery
• Prohance (2 mmol/l chelated Gadolinium) was added to the virus.
• Serial MRIs were acquired to monitor the infusate distribution within each target site and to provide real-time feedback to the surgical team. Animals were euthanized after 3 weeks and the brains were processed for immunohistochemical staining to assess the efficiency of distribution and transduction. Double fluorescence staining against the transgene, GFP, and neuronal marker, NeuN, was used to determine the percentage of transduced neurons (efficiency of transduction). 109.
• the results from the transduction of the non-human primate brain with LC.V1 showed that this vector is extremely efficient in the distribution and efficiency of transduction of large areas of the brain.
• the thalamus demonstrated near-complete coverage based on transgene expression (GFP) at the site of injection of only 124 ⁇ l of the LC.V1 vector ( Figures 4A-C).
• the average neuronal transduction efficiency from 4 thalamic nuclei was 67% (Figure 5I) as measured by GFP expression.
• LC.V1 is transported retrogradely from the site of injection (thalamus) to cortical regions where it transduces pyramidal neurons of prefrontal cortex and frontal cortex layer V ( Figures 4D-K and Figures 5A-C).
• LC.v1 This is significant as it supports the use of LC.v1 for treatment of disease affecting the prefrontal cortex.
• the neuronal transduction efficiency within the prefrontal cortex was 50% (Figure 5I) based on the average of 8 cortical regions, 4 lateral and medial, from 3 separate sections of brain tissue, all within Area 9 of prefrontal cortex.
• LC.V1 is transported retrogradely from the site of injection (thalamus) to the hippocampus and transduces neurons of the subiculum (Figures 5D-H).
• the average neuronal transduction efficiency within this structure is 53% (Figure 5I).
• LC.V1 is transported anterogradely from the site of injection (midbrain: VTA and substantia nigra) to the striatum (both caudate nucleus and putamen) where numerous GFP-positive fibers can be seen ( Figures 6A-D).
• Distribution of LC.V1 within the brain parenchyma delivered via CED can be monitored by real-time MRI imaging.
• ProHance gadoteridol
• the data shown herein also support the observation that LC.V1 is bidirectional showing both retrograde and anterograde transport. Also, we were able to monitor the vector distribution in real time. This is significant as an administering physician can monitor the vector administration and stop or adjust infusion when primary target is filled or being delivered to the correct location. 110.
• LC.V1 appeared to transduce neurons ( Figure 11) and interneurons in all the cortical and subcortical areas (pyramidal cell, medium spiny neurons, dopaminergic neurons, Purkinje etc).
• rAAV specifically AAV9
• LC.V1 performed at better distribution and higher levels of expression.
• LC.V1 When compared to AAV- PHP.B, we injected higher dose (1.0E+12 vg vs.4.18E+12 vg), LC.V1 performed at similar/better distribution and levels of expression. 111.
• animals C57BL/6 mice
• RO retro-orbital sinus
• TV Tail vein
• LV left lateral ventricle delivery
• Each animals received 50 ⁇ L systemically or 25 ⁇ L into the CSF of AAV:LC.V1 at a titer of 8.36E+13 vg/mL (lot# CS1851).
• Mol Ther 13 517-27 Lawlor PA, Bland RJ, Mouravlev A, Young D, During MJ.2009. Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates. Mol Ther 17: 1692-702 Liu X, Magee D, Wang C, McMurphy T, Slater A, During M, Cao L.2014. Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype.
• Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2.
• Nat Med 5 71-7 Rabinowitz JE, Samulski J.1998. Adeno-associated virus expression systems for gene transfer.
• Curr Opin Biotechnol 9 470-5 Rabinowitz JE, Xiao W, Samulski RJ.1999. Insertional mutagenesis of AAV2 capsid and the production of recombinant virus.

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  • 2021-09-03 EP EP21865201.4A patent/EP4208557A4/en active Pending
  • 2021-09-03 WO PCT/US2021/049081 patent/WO2022051633A2/en not_active Ceased
  • 2021-09-03 US US18/024,806 patent/US20230330267A1/en active Pending
  • 2021-09-03 CN CN202180054939.4A patent/CN116249541A/zh active Pending

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