Adeno-associated virus variant capsids and methods of use thereof
A mutation, virus technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc.
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[0092] Adeno-associated viruses (AAV) are a family of parvoviruses with a 4.7 kb single-stranded DNA genome contained within a non-enveloped capsid. The viral genome of naturally occurring AAV has 2 inverted terminal repeats (ITRs) - which function as the viral source of replication and packaging signals - flanked by 2 major open reading frames (ORFs): rep (encoded in the viral replication , transcription regulation, site-specific integration and virion assembly) and cap. The cap ORF encodes three structural proteins that assemble to form the 60-mer viral capsid. Many naturally occurring AAV variants and serotypes have been isolated, and none have been associated with human disease.
[0093]Recombinant versions of AAV can be used as gene delivery vectors, where a relevant marker or therapeutic gene is inserted between the ITRs replacing rep and cap. These vectors have been shown to transduce both dividing and non-dividing cells in vitro and in vivo, and can produce stable tr...
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[0291] The following examples are presented so as to fully disclose and describe to those of ordinary skill in the art how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all performed or single experiment. Efforts have been made to ensure accuracy with respect to quantities used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
[0292] General methods of molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Edition, (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Edition, (Ausubel et al., John Wi...
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[0294] Intravenous injection and tissue collection. For each round of selection, a single male cynomolgus monkey (cynomolgus monkey) aged 3-10 years and weighing at least 3 kg was administered intravenously via the great saphenous vein. Animals were anesthetized and dosed with 1-5 mL of the pool (in the first round, the pool was used by figure 1 Variant composition generated by all mutagenesis techniques described in A; in each subsequent round, variants were isolated from previous rounds), in some cases pre-incubated with human IVIG for 30 min at 37°C.
[0295] Euthanasia was performed by trained veterinary personnel using 100 mg / kg sodium pentobarbital intravenously on day 14±3 or 21±3, depending on choice. Cardiac and / or skeletal muscle tissue from the quadriceps is removed and DNA is isolated from the tissue. In some cases, myocardial tissue is divided into several regions: atrium, ventricular septum, left papillary muscle, right papillary muscle, left ventricle, and ri...
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