Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
1999-12-22
2002-11-12
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S320100
Reexamination Certificate
active
06479273
ABSTRACT:
The invention relates to a process for separating viruses of different sizes, with the virus-containing solution preferably being filtered through one or more filter membranes.
The original aim of gene therapy was to cure genetic diseases by altering body cells genetically in a suitable manner. Nowadays, the term gene therapy is extended to include genetically altered cells which can also be employed therapeutically for curing diseases which do not: have a genetic origin, such as viral diseases, for example.
The genetic alteration of therapeutically active cells requires suitable methods for transferring the nucleic acid, i.e. DNA or RNA, which brings about the genetic alteration of the cell. The nucleic acids which are to be transferred are frequently also described as being so-called transgenes, even when they do not exercise the functions of a gene, e.g. when they are anti-sense nucleic acids. In addition to the direct gene transfer of so-called naked nucleic acids, genetically altered viruses have also proved to be suitable for effecting the gene transfer. At present, retroviruses, adenoviruses or adeno-associated viruses (AAVs), inter alia, are being genetically altered so that they can each be used as a carrier (viral vector) of the transgene(s) for the gene transfer. An important consideration when developing suitable viral vectors is that of the safety aspects when using these vectors in gene therapy. In general, therefore, replication-deficient viruses are developed, that is viruses which, while being able to infect a cell and transfer the transgene(s) into the cell, are unable themselves to replicate in this cell. This is achieved, for example, by deleting the genes which are important for virus replication, for example the genes encoding structural proteins, and, where appropriate, incorporating the transgene(s) in place of them. The preparation of relatively large quantities, which are suitable for the use in gene therapy, of replication-incompetent viruses requires so-called helper viruses, which compensate, in the cell, for the defect in a replication-incompetent virus. The following examples of retroviral vectors and adeno-associated vectors are intended to clarify the general principle:
Replication-deficient retroviral vectors are derived from wild-type retroviruses, which constitute a separate family of eucaryotic viruses whose genetic material (structural genes and regulatory genes) is composed of single-stranded RNA. The viruses are composed of spherical, enveloped virus particles having a diameter of approx. 80-120 nm and an inner capsid, which contains two copies of the genomic RNA in the form of a ribonucleo-protein. For preparing retroviral vectors, one or more structural genes (gag, pol and/or env) is/are replaced by the transgene(s). The LTR (long terminal repeat) regions which are still present at the 5′ and 3′ ends contain, as cis-active elements, regulatory sequences such as a promotor, polyadenylation signals and the sequences which are required for integration into the genome. It is possible, therefore, for the retroviral vector only to contain the LTR regions which flank the transgene(s) The replication of a replication-deficient retroviral vector therefore requires, for example, a helper virus which contains one or more of the abovementioned retroviral structural genes and thus complements the deleted structural gene(s) (Whartenby, K. A. et al. (1995) Pharmac. Ther., 66, 175-190).
Replication-deficient adeno-associated viral vectors are derived from the wild-type AAV, which is a non-autonomously replicating representative of the parvoviruses and constitutes a single-stranded DNA virus having a diameter of approx. 25 nm. Today, it is possible to differentiate between the serologically distinguishable types AAV-1, AAV-2, AAV-3, AAV-4 and AAV-5. AAV viruses can either integrate into the genome of the host cell or replicate in the host cells in the presence of a helper virus. Adenoviruses were first of all found as possible helper viruses. In vertebrates, for example, adenoviruses form a group of more than 80 serologically distinguishable serotypes and contain an outer, icosahedral protein coat (capsid) and an inner, central DNA-protein body (core). The capsid is in turn composed of 252 subunits, so-called capsomers. In general, the adenoviruses have a diameter of approx. 70-90 nm and contain, as the genetic material a double-stranded, linear DNA at the 5′ and 3′ ends of which there are ITR regions (inverted terminal repeats). AAV replication (lyric phase) now requires, in particular, the expression of early adenoviral genes such as, e.g., the E1a, E1b, E2a and E4 genes and the VA RNA (Kotin, R. M. (1994) Human Gene Therapy, 5, 793-801). However, other helper viruses, such as the herpesviruses, which is a group of double-stranded DNA viruses which are pathogenic to humans and animals and which have a diameter of approx. 120-200 nm, with the capsid having an icosahedral structure and being composed of 162 capsomers, are also suitable. The herpesviruses can be divided into three subfamilies, with, for example, type I and type 2 herpes simplex viruses (HSV) belonging to the Alphaherpesvirinae, cytomegalovirus (CMV), for example, belonging to the Betaherpesvirinae, and Epstein Barr virus (EBV), for example, belonging to the Gamma-herpesvirinae.
In analogy with the retroviral vectors, one or more of the rep genes which are required for replication (e.g. rep 40, rep 52, rep 68 and/or rep 78) or the cap genes which are required for the capsid structure (e.g. VP-1, VP-2 and/or VP-3) can, for example, be replaced with the transgene(s) when preparing adeno-associated vectors. The ITR regions which are still present at the 5′ and 3′ ends are needed, as cis-active elements, for packaging the transgene into infectious, recombinant AAV particles and for the replication of the DNA of the recombinant AAV genome (Kotin, R. M. (1994), loc. sit.).
Cotransfection of a eucaryotic cell with two recombinant AAV plasmids and a helper virus (Chiorini, J. A. et al. (1995) Human Gene Therapy, 6, 1531-1541) is an advantageous method for preparing relatively large quantities of recombinant AAV particles. The first recombinant AAV vector contains the transgene(s) which is/are flanked by the two ITR regions. The second recombinant AAV plasmid contains the AAV genes which are required for preparing the particles (rep and cap genes). The absence of functional ITR regions in the second vector prevents the rep and cap genes being packaged into AAV particles and undesirable wild-type AAV thus being formed. Mammalian cells, for example COS-7 cells, which are permissive both for the recombinant AAV vectors and for the helper virus, for example adenovirus, i.e. which provide the prerequisites for infection and replication, are then transfected with the two recombinant AAV vectors and the helper virus. The adenovirus is particularly suitable for use as the helper virus since it can infect a broad spectrum of target cells and can replicate in the cells themselves. When the transfected cells are cultured, the AAV non-structural protein genes and the AAV structural protein genes are expressed, the transgene DNA is replicated and the recombinant AAV particles (rAAV particles) are packaged and assembled. The rAAV particles contain the transgene(s), which is/are flanked at both ends by the ITR regions, in the form of single-stranded DNA. At the same time, the helper virus replicates in these cells, something which generally ends, when adenoviruses are used as helper viruses, in the lysis and death of the infected cells after a few days. The resulting viruses (adenoviruses and rAAV particles) are either in part released into the cell culture supernatant or else remain in the lysed cells. For this reason, the cells are generally disrupted using cell disruption methods which are known to the skilled person, such as alternately freezing and thawing or by means of enzymic hydrolysis, for example with trypsin (Chiorini, J. A. et al. (1995), loc. sit.), in order to
Bogedain Christoph
Hörer Markus
Maass Gerhard
Clark & Elbing LLP
MediGene Aktiengesellschaft
Mosher Mary E.
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