Compositions and methods to prevent AAV vector aggregation

A kind of composition, the technology of virus particle, be applied in the composition field of AAV virus particle, can solve the problem such as high cost and output limitation

Inactive Publication Date: 2007-08-15
GENZYME CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vector aggregation leads to loss during vector purification, and while aggregates can be removed by filtration, yield loss leads to high cost and yield limitations when producing vectors for preclinical and clinical research

Method used

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  • Compositions and methods to prevent AAV vector aggregation
  • Compositions and methods to prevent AAV vector aggregation
  • Compositions and methods to prevent AAV vector aggregation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] AAV purification method

[0074] AAV2 vectors expressing human coagulation factor IX (FIX) or human amino acid decarboxylase (AADC) were generated by three transfections of HEK293 cells as previously described (Matsushita, T. et al. (1998) Gene Therapy 5:938-945) , improvements were made. For large-scale preparations, the 850mm 2 Cells were cultured and transfected in shake flasks (Corning). Vectors were purified by one of three methods.

[0075] In purification method 1, modified from Matsushita, transfected HEK293 cells in shake flasks were harvested by centrifugation (1000 g, 15 minutes), resuspended in 10 mM sodium phosphate, 500 mM sodium chloride, pH 7.2, and frozen by three times. / thaw cycle to lyse (alternative ethanol / dry ice bath and 37°C water bath). Cell lysates were clarified by centrifugation (8,000 g, 15 minutes). The supernatant was then diluted to 200 mM NaCl by adding 10 mM sodium phosphate, pH 7.2 and treated with Benzonase (Merck, purity grad...

Embodiment 2

[0081] Ultrafiltration and Diafiltration to Detect AAV Aggregation

[0082] Disposable hollow fiber tangential flow filtration devices (Amersham BioSciences 8" Midgee, 100 kDa nominal pore size) were used to concentrate and diafilter AAV2 vectors purified by the method described above and for the UF / TF experiments described in Table 2 For all UF / DF procedures, use a volume of diafiltration buffer corresponding to 10 x product volume and add to near continuous diafiltration in ~1 mL increments. Using this method, the calculated residual CsCl after diafiltration < 0.5mM.

[0083] The following three formulations were used for UF / DF: Control formulation (CF: 140 mM sodium chloride, 10 mM sodium phosphate, 5% sorbitol, pH 7.3); Test formulation 1 (TF1: 150 mM sodium phosphate, pH 7.5) and Test Formulation 2 (TF2: 100 mM Sodium Citrate, 10 mM Tris, pH 8.0). For Experiment 1 shown in Table 2, the corresponding 1×10 13 The volume of vg / mL carrier concentration was diafiltered, and...

Embodiment 3

[0087] Measuring Carrier Aggregation by Dynamic Light Scattering

[0088] Aggregation of purified vectors was analyzed by dynamic light scattering (DLS) using protein solution DynaPro99 (λ = 825.4 nm). Raw data (particle radius - Rh, mean value measured over 30 cycles, 10 cycles / min) were used for all analyzes reported. A "dilution-stress" approach was used to test the effect of various excipients on carrier aggregation. In this method, 80 μL of test diluent is added to 20 μL of vehicle solution and mixed in an actual cuvette for DLS measurement, and data collection begins within 10 seconds of mixing. Before adding the test diluent, the Rh value of the AAV2 vector formulation was measured and confirmed to be <15nm to ensure that the starting material was monomeric. Samples that were not 100% monomeric were passed through a 0.22 μm syringe disc filter (Sartorius, low protein binding) to remove aggregates.

[0089] The osmolality and ionic strength given in Figures 1 and 2 we...

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Abstract

Compositions and methods are provided for preparation of concentrated stock solutions of AAV virions without aggregation. Formulations for AAV preparation and storage are high ionic strength solutions (e.g. mu~500mM) that are nonetheless isotonic with the intended target tissue. This combination of high ionic strength and modest osmolarity is achieved using salts of high valency, such as sodium citrate. AAV stock solutions up to 6.4x10<13> vg / mL are possible using the formulations of the invention, with no aggregation being observed even after ten freeze-thaw cycles. The surfactant Pluronic(R) F68 may be added at 0.001% to prevent losses of virions to surfaces during handling. Virion preparations can also be treated with nucleases to eliminate small nucleic acid strands on virions surfaces that exacerbate aggregation.

Description

technical field [0001] The present invention relates to compositions and methods for preparing and storing AAV virions that prevent aggregation. Background technique [0002] Recombinant adeno-associated virus (rAAV) is a promising vector for human gene transfer. Grimm, D. and Kleinschmidt, J.A. (1999) Hum Gene Ther. 10:2445-2450; High, K.A. (2001) Ann.N.Y.Acad.Sci.953:64-67; Pfeifer, A. and Verma, I.M. (2001 ) Ann. Rev. Genomics Hum. Genet. 2:177-211. AAV is a member of the genus Dependovirus in the family Parvoviridae. AAV serotype 2 (AAV2) consists of a 4680 nucleotide single-stranded DNA molecule encoding a replication (rep) and encapsidation (cap) gene flanked by inverted terminal repeat (ITR) sequences. Bers, K.I. (1996) Fields Virology (eds. B.N. Fields et al.), pp. 2173-2197. Lippincott-Raven Publishers, Philadelphia. The genome is packaged by three capsid proteins (VP1, VP2 and VP3), which are amino-terminal variants of the cap gene product. The resulting icos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02
Inventor J·F·赖特G·曲
Owner GENZYME CORP
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