JP2002516345A - Adeno-associated virus vector-mediated expression of factor VIII activity - Google Patents

Abstract

  (57) [Summary] The present invention provides methods and materials for expressing a polypeptide having Factor VIII activity. The method comprises administering different domains of human factor VIII and at least two rAAV vectors encoding at least a heavy and light chain.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION I. Adeno-Associated Virus Adeno-associated virus (AAV) is a defective parvovirus whose genome is encapsulated as a single-stranded DNA molecule in a capsid. The positive and negative polarity strands are packaged in separate virions with equal efficiency, but with different efficiencies. AA
Efficient replication of V usually requires co-infection with a helper virus of the Herpesviridae or Adenoviridae family. However, under certain circumstances, AAV can replicate in the absence of a helper virus.

In the absence of a helper virus, AAV establishes a latent infection in which the viral genome is present as an integrated provirus in a host cell. Virus integration occurs on human chromosome 19. If the latently infected cell line is later superinfected with the appropriate helper virus, the AAV provirus is generally excised and the AAV virus enters a "growth" phase.

[0003] AAV isolates have been obtained from humans and simians. AAV
The host range for lytic growth of E. coli is extensive. Cell lines from virtually any mammalian species to be tested, including various human, monkey, canine, bovine and rodent cell lines, are suitable helper viruses (eg, canine adenovirus in canine cells). If) is used, it can be productively infected with AAV. However, there is no disease associated with AAV in either humans or other animal populations.

[0004] AAV has been isolated as a non-pathogenic co-infectious agent from fecal, ocular and respiratory specimens during acute adenovirus infection, but not from specimens from other diseases. Latent AAV infections have been identified in both human and non-human cells. In general, viral integration does not appear to have any apparent effect on cell growth or morphology. Samulski, Curr. Op. Gen. Level
. , 3: 74-80 (1993).

[0005] There are a number of AAVs, including AAV-1, AAV-2, AAV-3, AAV-4, and AAV-5. The AAV-2 genome is 4,679 bases long (Genb
ank, number AF043303), and each contains an inverted terminal repeat of 145 bases. This repetition is thought to serve as an origin of DNA replication. A
The AV-3 genome is 4726 bases in length and has 82% overall sequence homology to AAV-2. Muramatsu, Virology, 221: 20
8-217 (1996). Like AAV-2, both ends of the AAV-3 genome consist of inverted repeats, but the palindrome is 146 bp in size. A
Certain parts of the AV-2 and AAV-3 genomes are highly conserved, for example, the hairpin has two sites with only one base pair substitution between AAV-2 and AAV-3. Exists.

[0006] The AAV genome has two major open reading frames. The left frame encodes at least four nonstructural proteins (Rep group).
There are two promoters, P5 and P19, that control the expression of these proteins. As a result of various splicing, the P5 promoter directs the production of proteins Rep78 and Rep68, and the P19 promoter directs the production of proteins Rep52 and Rep40. Rep protein is
It is thought to be involved in viral DNA replication, transactivation of transcription from viral promoters, repression of heterologous enhancers and promoters, and site-specific integration.

The right ORF controlled by the P40 promoter is the capsid proteins Vp1 (91 kDa), Vp2 (72 kDa) and Vp3 (60 kD
Code a). Vp3 contains 80% of the virion structure, while Vp1 and Vp2 contain few components. The map unit 95 has a polyadenylation site. For the complete sequence of the AAV-2 genome, see Strivastava.
J. et al. Virol. 45: 555-64 (1983).

[0008] McLaughlin et al. Virol. , 62: 1963-73 (198
8) prepared two AAV vectors: dl 52-91 (this is the AAV
Dl gene, and dl 3-94 (here, all AAV
Coding sequence has been deleted). However, dl 3-94 retains two 145 base terminal repeats, and an additional 139 bases, including the AAV polyadenylation signal. A foreign gene (encoding neomycin resistance) was inserted into the vector. A viral stock was prepared by complementing with a recombinant AAV genome that trans-supplied the missing AAV genomic product, but was itself too large to be packaged. Unfortunately, the viral pool was contaminated with wild-type AAV (10% in the case of dl 3-94), probably as a result of homologous recombination between the defective and complementary viruses.

[0009] Samulski et al. Virol. 63: 3822-28 (1989) developed a method for producing a recombinant AAV depot without detectable wild-type helper AAV. The AAV vector retained only the terminal 191 bases of the AAV chromosome. In the AAV helper plasmid (pAAV / Ad), the terminal 191 bases of the AAV chromosome were replaced with adenovirus terminal sequences. in this way,
No detectable wild-type AAV was generated by homologous recombination because sequence homology between the vector and the helper AAV was essentially eliminated. In addition, the helper DNA itself was not replicated and not encapsulated. Because the process requires an AAV end. Thus, in this AAV system, unlike the HSV system, the helper virus is completely eliminated and a helper-free AAV vector stock can be generated.

[0010] Recombinant AAV (rAAV) vectors have been used for gene product expression in animals. For example, US Pat. No. 5,193,941 and WO 94 /
See 13788. Other patents and publications describe AAV vectors and uses. Here, this use is generally performed in vitro (usually in tissue culture) or in vivo (usually in the lung or oral mucosa, which is a normal site of AAV infection).
However, expression in other tissues such as the central nervous system and heart tissue has been observed).

[0011] Transduction of the rAAV vector carrying the bacterial β-galactosidase gene by a single injection into the quadriceps of mice demonstrated that expression was maintained for an extended period and expression was not substantially reduced during that time. (Xiao et al., J. Virol., 70:
8098-8108 (1996)). Other targets successfully transduced with the rAAV vector include: T-lymphocytes and B-lymphocytes, human erythroleukemia cells, different regions of rat brain, rats in a Parkinson's disease model using the tyrosine hydroxylase gene. Brain striatum, pig and rat hearts with LacZ gene, guinea pig peripheral auditory system, and rabbit and monkey bronchial epithelium. In addition, human Phase I clinical trials for delivery of the rAAV-CFTR construct are ongoing.

AAV carrying the human IX gene was injected into the portal vein of adult immunoreactive mice and long-term gene expression was obtained (Snyder et al., Nat. Gen.
et. 16: 270-276 (1997)). This vector was found to integrate into the mouse genome (Miao et al., Nat. Genet., 19:
13-15 (1998)).

II. Hemophilia Hemophilia A is a component of the coagulation cascade, Factor VIII (FVIII)
Or X chromosome-related bleeding disorders resulting from fVIII) deficiency or abnormality. The human FVIII cDNA has been cloned. FVIII is 19
235 composed of amino acid signal peptide and 6 different domains
Synthesized as a single-chain precursor of one amino acid residue. This domain is in turn A1
-A2-B-A3-C1-C2 (Toole et al., Nature, 3
12: 342-347 (1984); Vehar et al., Nature, 312: 3.
37-342 (1984)). The A domain contains about 330 amino acids and there are three copies. The C domain contains about 150 amino acids and there are two copies. The B domain contains about 909 amino acids and is highly enriched for potential N-linked glycosylation sites.

The translation product of the FVIII gene is first cleaved between the B and A3 domains. The B domain is then proteolytically digested at multiple sites and 90-20
Releases FVIII as a divalent metal ion binding complex consisting of a 0 kDa heavy chain (H chain) and an 80 kDa light chain (L chain) (Vehar et al., (1984), supra; Anderson et al., Proc. Natl. Acad.Sci.USA, 8
3: 2979-2983 (1986)).

[0015] The minimal functional unit of FVIII is a heterodimer consisting of a 90 kDa heavy chain and an 80 kDa light chain. Thus, the B domain is unnecessary for procoagulant activity (Eaton et al., Biochemistry, 25: 8343-
8347 (1986); Toole et al., Proc. Natl. Acad. Sci
. USA 83: 5939-5942 (1986)). Circulating F in the blood
VIII is associated with a large multimeric multifunctional product, von Willebrand factor (vWF) (Brinkhouse et al., Proc. Natl. Ac).
ad. Sci. ScL USA, 82: 8752-8756 (1985)).

[0016] Hemophilia A patients are at risk for developing an infectious disease transmitted from the plasma-derived FVIII used in the treatment. Thus, recombinant products are a desirable alternative. However, the complex processing and large size of FVIII has prevented production of FVIII in prokaryotes or lower eukaryotes.

Although expression of full-length FVIII cDNA in mammalian cells has been reported by several groups, the level of expression is very low and recombinant FVIII
(RFVIII) was insufficient for economical production.

Modified FVII lacking most of the B domain to improve expression efficiency
I cDNA was generated (Eaton et al., (1986), supra; Toole et al., (1986), supra; Sarver et al., Behring Inst. Mitt.
. , 82: 16-25 (1988); Meulien et al., Protein En.
gng. , 2: 301-306 (1988); Tajima et al., Proc. 6t
h, Int. Symp. , II. T: 51-63 (1990)), and the resulting product has been shown to retain FVIII functional activity.

(1990), supra, fused the coding sequences for the heavy and light chains. The construct was expressed about 10-fold higher than the full-length FVIII cDNA construct, but 20% of the product was not cleaved into heavy and light chains or was cut incorrectly. Eaton et al. ((1986), supra) inserted a connecting peptide from the B domain between the heavy and light chains. However, this connecting peptide remained at the C-terminus of the H chain. Such fusion molecules have antigenic properties (Esmon et al.,
Blood 76: 1593-1600 (1990)). This can cause severe side effects due to the constant exposure of the host to these antigens during long-lasting treatment.

Burke et al. (J. Biol. Chem., 261: 12574-12578).
(1986)) has a heavy chain (Ala1 to Arg740) and a light chain (Glu164).
9-Tyr2332) was expressed as another protein in COS cells, and secretion of functionally active FVIII was observed. However, this expression level was even lower than that of the full-length construct. Yonemura et al. (Prot.
Enng. , 6: 669-674 (1993)) basically used two plasmids to deliver heavy and light chain genes into CHO cells.

Another complication of the disease is the observation that the severity of the bleeding tendency varies between patients and may be related to the level of functional coagulation factors. Individuals may have mild hemophilia that may not be recognized until adulthood or after severe trauma or surgery. See, for example, Reiner and Davier, "Introductory.
ion to hemostasis and the vitamin K-
"Dependent Coagulation Factors" The Me
tabolic Basis of Inherited Disease (S
Criver et al., Ed., Vol. 3, pp. 3181-221 (McGraw Hill)
, New York, 1995).

III. Therapy Adenoviral vectors can infect non-dividing cells and can therefore be delivered directly to mature tissues such as muscle. However, transgenes delivered by adenovirus vectors are not useful for long-term expression for various reasons. First, adenoviral vectors carry most of the viral genes and thus pose a potential problem (ie, safety). Expression of adenovirus genes can cause the immune system to destroy cells containing this vector (see, eg, Yang et al., Proc. Na.
tl. Acad. Sci. , 91: 4407-4411 (1994)). Since adenovirus is not an integrating virus, this vector is ultimately diluted or degraded in cells. Also, due to the immune response, adenovirus vectors cannot be delivered repeatedly. In the case of a permanent disease such as hemophilia, this is a significant limitation.

For a retroviral vector, stable integration into the host chromosome is achieved, but its use is limited. Because currently used vectors can only infect dividing cells, most of the target cells are not dividing.

AAV vectors have particular advantages over the above vector systems. First,
Like adenovirus, AAV infects nondividing cells. Second, all AAV viral genes are eliminated in the vector. AAV vectors are safer than adenovirus vectors because the viral gene expression-induced immune response is no longer involved. Since AAV is an integrating virus, integration into the host chromosome maintains the transgene in the cell. AAV contains many surfactants, p
It is a very stable virus that is resistant to H change and heat (stable at 56 ° C for about 1 hour)
. AAV can be lyophilized and redissolved without significant loss of activity. Finally, AAV produces no known disease or pathogenic symptoms in humans. Therefore, A
AV is a very promising delivery vehicle for gene therapy.

Two recent review articles provide a review of recent situations in the use of AAV vectors and include a collection of more recent scientific publications in the field:
Samulski, "Viruses in Human Gene Therapy", edited by Vos, Chapman and Hall, 1994, "Aden
o-associated Viral Vectors ", Chapter 3, and" G.
ene Therapy: From Laboratory to the C
linic ", edited by Hui, S in World Scientific, 1994.
amulski, "Adeno-associated Virus-base"
d Vectors for Human Gene Therapy ", 11
chapter.

AAV has in the past a broad host range; can be administered by a variety of routes, including intramuscular injection; and can be manipulated in different cell types (eg, liver, retina, nerve, etc.) Is shown, the host on which the delivery methods described herein can be performed, in particular, for example, mammals, especially domestic mammals (eg, cows, sheep, pigs, horses, dogs) , Cats, chickens and turkeys).

SUMMARY OF THE INVENTION The present invention demonstrates for the first time that recombinant AAV vectors can be used to deliver effective expression of proteins with Factor VIII function and treat hemophilia A.
Multiple vectors provide the host cell with FVI for expression and acquisition of FVIII activity.
Deliver the II domain. Methods described herein, and recombinant AA
V vectors offer an important development in the field of recombinant AAV vector gene therapy.

The methods and materials of the present invention provide significant advantages. This advantage includes FVII
An rAAV vector carrying a gene for expressing a molecule having I activity at a high level can be delivered to an intact animal via a route of delivery, such as, but not limited to, intraportal or intravenous administration, eg, The ability to deliver to liver or endothelial tissue is included.

The rAAV vector of the present invention can be used as a nucleic acid or a virus particle.
Alternatively, rAAV vector viral particles can be used alone or in combination with further treatments. This treatment includes partial hepatectomy or treatment with a second agent that enhances transduction, whether or not related to in vivo or ex vivo therapy. Examples of secondary agents include gamma gland irradiation, UV irradiation, tritiated nucleotides (eg, thymidine), cis-platinum, etoposide, hydroxyurea, aphidicolin, and adenovirus.

Description of Particular Embodiments FVII Related to FVIII Precursor Size and AAV Vector Size
Due to the difficulties in cloning and expression of I and the extensive processing difficulties of its precursor, at least the light (L) chain containing the A domain and the two C domains and the heavy (H) chain containing the two A domains VII as long as it is expressed
Multiple A, each carrying one or more FVIII domains to obtain factor I activity
It has been discovered that AV vectors can be used. The plurality of AAV vectors
Despite the phenomenon of viral immunity, they can infect single cells or different cells. A suitable approach is to use two vectors, one carrying the sequence encoding the FVIII heavy chain (HC) and the other carrying the sequence encoding the FVIII light chain (LC). is there. However, it is understood that other modifications are possible.

[0031] The rAAV vector of the present invention is a derivative of an adeno-associated virus into which a factor VIII sequence having various modified sequences has been introduced.

The wild-type adeno-associated virus is depleted when it requires helper cells for lytic infection, but the subject to which the vector is delivered recruits the rAAV vector and produces rAAV particles. There is a possibility to carry a herpesvirus or adenovirus infection that can lead. To protect against that possibility, rAA
Modify the V vector to reduce the likelihood of rescue. In theory, such modifications may take the form of point mutations in one or more viral genes. This mutation either prevents expression of the entire gene or results in the expression of a non-functional modified gene product. However, point mutations can be reversible. Consequently, it is preferable to simply delete each unwanted adeno-associated virus gene, and this creates more space within the viral package of larger nucleic acids containing FVIII sequences and / or regulatory elements. Has further advantages.

[0033] All viral genes may be deleted from the rAAV vector or otherwise known AAV vector d13-94 (see, eg, McLaughlin, J. et al.
Virol. , 62: 1963-1973 (1988)). However, rAAV vectors carrying one or more AAV genes (eg, the known AAV vector d152-91) are still likely to be inferior to preferred vectors containing non-functional AAV genes,
It should be appreciated that it may be useful for gene delivery; Hermonat,
J. Virol. , 51: 329-339 (1984). Preferably, AAV-derived rAAV vectors essentially retain only recognition signals for replication and packaging (ITR).

For in vitro propagation of the rAAV vector, the susceptible cells are the AAV-derived vector DNA and the appropriate AAV-derived helper virus, or AAV.
It is co-transfected with a plasmid carrying the rep gene, the AAV cap gene or both, and infected by a helper virus, including a herpes virus, an adenovirus or an appropriate helper plasmid. The particular method of producing the virus particles is not critical to the invention. As long as appropriate concentrations of virus particles capable of transducing cells in vivo and ex vivo are obtained, Sam
Any method of producing rAAV virions, including but not limited to those described in ulski et al. (1989), supra, may be used. One skilled in the art will understand that any purification method used should produce infectious viral particles that can, for example, transduce hepatocytes or endothelial cells in vivo or ex vivo.

It is not necessary that the AAV-inducing sequence correspond exactly to the wild-type AAV prototype. For example, a rAAV vector of the invention can be modified inverted terminal repeats and other rAAV vectors, provided that the rAAV vector replicates and is packaged with the helper virus and can establish a non-pathogenic latent infection in target cells. The sequence can be characterized.

The exact nature of the regulatory regions required for gene expression can vary from organism to organism, but generally includes a promoter that directs the initiation of transcription of RNA in the cell of interest. Such regions contain 5 ′ non-coding sequences involved in transcription initiation (eg, TATA).
Box). The promoter can be constitutive or regulated. A constitutive promoter is a promoter that essentially causes a operably linked gene to be expressed at any time. A regulatable promoter is a promoter that can be activated or deactivated. Regulated promoters include inducible promoters, which are usually "off" but can be induced to be "on", and "repressible" promoters are usually "on", "Off".
Many different regulators are known, including temperature, hormones, cytokines, heavy metals and regulatory proteins. These distinctions are not absolute, and constitutive promoters can be regulated to some extent.

The regulation of a promoter can be associated with a particular genetic element to which an inducer or repressor binds, often called an “operator”. The operator can be modified to change its adjustment. Hybrid promoters can be constructed such that the operator of one promoter is moved to another.

A promoter is a “ubiquitous” promoter (eg, the β-actin or cytomegalovirus promoter) that is active in essentially all cells of the host organism.
Or a promoter with expression that is more or less specific for the target cell (eg, an albumin promoter). Preferably, a tissue-specific promoter is, for example, essentially inactive outside the hepatic system, and the activity of the promoter is optionally higher in some components of the hepatic system than in other systems. possible.

Thus, the promoter may be a promoter that is mainly active in the hepatic system. This specificity can be absolute or relative. Similarly, the promoter may be specific for a particular cell type, including but not limited to hepatocytes, Kupfer cells or endothelial cells.

In general, to determine the location of a tissue-specific promoter, a gene that is expressed only (or predominantly) in that tissue is identified, and then the gene encoding the protein is isolated. (The gene may be a normal cellular gene or may be a viral gene of a virus that infects the cell.) The promoter of that gene, when linked to another gene, provides the desired tissue-specific activity. It seems to hold. The tissue specificity of the promoter may be associated with a particular genetic element, which may be modified or transferred to a second promoter.

One skilled in the art will recognize that tissue-specific promoters for use in AAV vectors are the albumin promoter; the alpha-fetoprotein promoter (Genbank accession number L34019); the alpha-fetoprotein enhancer; the human apolipoprotein E (ApoE) gene promoter and its associated liver. Specific enhancers HCR-1 and HCR-2 (Nguyen et al., Oncogene, 12 (1
0): 2109-2119 (1996) and Allen et al. Biol. C
hem. 270 (44): 26278-26281 (1995));
Factor I promoter (Genbank accession number M14113); Factor IX promoter (Sailer et al., (1990), below); vWF promoter (Gn
Atenko et al., Blood, 90 Addendum, Part 1: 119a (1997).
Liver-specific enhancer of apolipoprotein AI (Malik et al., Mol)
. Cell. Biol. , 16 (4): 1824-1831 (1996))
Factor III promoter, Factor IX promoter, vWF promoter and liver-specific human α1-antitrypsin promoter (Wu et al., Hum. Gene.
Therapy, 7 (2): 159-171 (1996) and Hafenri.
chter et al., Blood, 84 (10): 3394-3404 (1994)).
May be selected from any known liver-specific promoters and enhancers, including

There are also other known strong promoters that find general use for obtaining high levels of recombinant protein expression. For example, the herpes simplex thymidine kinase promoter, SV40 promoter and LTR have found widespread use as strong promoters. An example is the LTR obtained from Moloney leukocyte retrovirus.

For the gene to be expressed, the coding sequence must be operably linked to a promoter sequence that is functional in the target cell. If the promoter is activated such that the coding sequence is transcribed when the promoter is activated, the promoter region is operably linked to the coding sequence. If the ligation does not result in an error in reading the downstream sequence, the coding sequence is operably linked. "
To be "operably linked", the two sequences need not be immediately adjacent to one another.

[0044] The rAAV vector may further include one or more restriction sites at which polynucleotides carrying the FVIII domain (s) can be cloned without interfering with packaging and replication. Preferably, at least one unique restriction site is provided. rAAV vectors may also include one or more marker genes to facilitate genetic manipulation. Suitable marker genes are known in the art and include, but are not limited to, neomycin and hygromycin resistance genes, bacterial lacZ and firefly luciferase genes.

For any mRNA slice donor / splice acceptor sequence associated with an intervening sequence, any such donor / acceptor sequence compatible with and operable in the AAV can be used. Certain non-limiting examples of suitable donor / acceptable sites are taught herein and are known in the art.

With respect to the polyadenylation site, any such polyadenylation site that is compatible with and operable in the AAV can be used. Specific, non-limiting examples of suitable polyadenylation sites are taught herein, and all are known in the art.

If desired, a non-coding region 3 ′ to the gene sequence encoding the desired RNA product can be obtained. This region may be retained for its transcription end regulatory sequences, such as those that provide for termination and polyadenylation. Therefore, FVIII
Retaining the 3 'region naturally adjacent to the coding sequence of the light chain can provide a transcription termination signal. If the transcription termination signal that naturally associates with the coding sequence does not function well in the expression host cell, a different 3 'functional in the host cell.
Regions can be replaced.

Regarding the AAV inverted terminal repeat, any of its AAV vectors carrying foreign genes, including any nucleotide substitutions, deletions, inversions and / or insertions, that still retain the required essential biological activity of the AAV vector. Such an ITR or derivative thereof may be used. ITRs from different AAV serotypes can also be used.

Another element that contributes to the proper expression of FVIII is a signal sequence.
A suitable signal sequence is the sequence of native FVIII. Thus, the native FVIII signal sequence is cloned upstream of the N-terminal amino acid of the FVIII domain sequence in multiple vector constructs.

Typically, due to the packaging limitations of AAV, polynucleotides encoding FVIII domain sequences and regulatory sequences can be up to about 5,500 bases in length.

The AAV helper virus or helper plasmid is the AAV that it carries.
Upon expression of the gene, it can be any virus or plasmid that can provide the proteins required for replication and packaging of the rAAV vector in a suitable host cell for the purpose of generating a stock of the rAAV vector. .

A non-AAV helper virus or non-AAV helper plasmid has been engineered to reduce the risk of recombination between non-AAV helper DNA and rAAV vector DNA.

Therefore, it is preferred that the sequence homology between the AAV sequence of the vector DNA and the AAV sequence of the helper DNA is very limited or absent. For example, the helper DNA contains AAV inverted terminal repeats that have the corresponding sequence of another virus (
For example, it may be an AAV sequence that has been replaced by adenovirus type 5 DNA (see Samulski et al. (1989) supra).

The non-AAV helper virus may be a virus in which a gene required for replication has been deleted. For example, an adenovirus stock in which one gene of the adenovirus (eg, the EIA gene) has been disabled is desirable.

Any of a variety of methods, including heat inactivating the non-AAV helper virus at about 56 ° C. for about 30-45 minutes, or performing single or multiple centrifugations in cesium chloride. By the method, it can be removed by physically separating it from the packaged rAAV vector.

The basic procedure for the construction of recombinant DNA and RNA molecules according to the present invention is described in numerous publications (Sambrook et al .: Molecular Cloning: A
Laboratory Manual, 2nd edition, Cold Spring Ha
rbor Press, Cold Spring Harbor, NY (198
9), which is incorporated herein by reference).

The present invention can be used for gene therapy of a factor VIII-related disorder (eg, hemophilia A). As a result of one or more mutations in the regulatory region and / or coding sequence of the Factor VIII gene, Factor VIII is inappropriately expressed (eg, having an incorrect amino acid sequence, in the wrong tissue, or Expressed at an early stage, underexpressed, or overexpressed)
For that reason, some individuals may require gene therapy.

The target cells of the vectors of the present invention may be cells capable of expressing a polypeptide having FVIII activity (eg, cells of the mammalian hepatic system, endothelial cells and proteins having factor VIII activity). Other cells that have the proper machinery to process secreted precursors). In one embodiment, the cells are normal cells cultured in vitro. Target cells can be human cells, or cells of other mammals, such as non-human primates, rodents, carnivores and other livestock species.

[0059] In another embodiment, the cells are delivered when the rAAV vector is delivered to the cells.
Part of a living mammal. The mammal can be at any stage of development at the time of delivery, such as an embryo, fetus, infant, young, or adult. Further, the cells can be healthy or pathological.

The vector can be administered by intraportal injection, for example, to specifically deliver to a particular region of the liver. Since AAV vectors are stably retained in target cells (rather than producing viral particles), subsequent propagation of the vector is small and is primarily an effect of passive diffusion from the injection site. The degree of diffusion can be controlled by adjusting the ratio of rAAV vector to liquid carrier.

[0061] In certain embodiments, the vector is administered via an intravascular approach. For example, the vector can be administered intra-arterially. Of course, for intravenous and intraportal delivery, the recipient mammal must be able to withstand the potential for delivery of the vector to cells other than cells of the hepatic system.

To target a vector to a particular cell type (eg, hepatocytes), it may be beneficial to associate the vector with a homing factor that specifically binds to cell surface receptors. Thus, the vector can be conjugated to a ligand (eg, galactose), and certain hepatic cells have receptors for that ligand. The linkage can be covalent (eg, using a cross-linking agent (eg, glutaraldehyde)) or non-covalent (eg, binding of an avidinated ligand to a biotinylated vector). Another form of covalent linkage is where one or more of the encoded coat proteins is a hybrid of a native AAV coat protein and a peptide or protein ligand, such that the ligand is exposed to the surface of the viral particle. Provided by designing an AAV helper plasmid using vector stock preparation.

No form of ligation should substantially interfere with either production or transformation of the rAAV vector.

The use of multiple rAAVs to transform a single cell may depend on whether the host cell is polynuclear, polysomy or polyploid. Thus, other suitable target cells can be syncytia cells, muscle cells, germ cells, bone marrow cells, megakaryocytes, and the like. However, the invention is not to be construed as limited to such cells, and as long as it results in proper processing and expression of a polypeptide having Factor VIII activity, it is normally diploid, in somatic cells Can be similarly performed on mononuclear cells.

[0065] As used herein, a multinucleated cell is a cell that contains one or more nuclei. A polynuclear cell can be all or part of a polyploid or polysomal cell. The development of one or more nuclei in a cell is not necessarily dependent on having multiple complete diploid sets of chromosomes. However, often multinucleated cells contain multiple complete diploid sets of chromosomes.

The rAAV vector was transformed with the rAAV vector virus particles in vitro, either as direct delivery in vivo to the portal vasculature or into cells from treated animals, and then transformed into donors. Either as an ex vivo treatment involving the introduction of cells back, they can be administered alone as viral particles. Alternatively, rAAV vector virions are used in combination with a second factor known to enhance transformation efficiency (see, for example, WO 95/33824 using any of a variety of second factors). Can be used to transform cells. Second factors useful for enhancing transformation efficiency include radioactive molecules, including: tritiated nucleotides, ultraviolet radiation, gamma irradiation, cisplatin, hydroxyurea, etoposide, camptothecin, aphidicolin, and adenocholine. Viruses (e.g., Ferrari et al., J. Virol. 70: 3227-).
3234 (1996)).

Prior to administration to a host, it is beneficial to determine the purity of the recombinant AAV preparation, ie, the AAV vector contained in the transgene. For example, the disease is A
Although not associated with AV infection, it is desirable to know the extent of wild-type AAV contamination in recombinant virus preparations. The wild-type virus is, for example, a crossover between the AAV helper plasmid and the AAV plasmid having the transgene.
).

The presence of contaminating wild-type AAV may be, for example, a nucleic acid amplification assay (eg, PCR
) Or RNA-based amplification methods (eg, 3SR (Gingeras et al., An)
n. Biol. Clin. 48: 498-501 (1990) and NASB.
A (van der Vleit et al., J. Gen. Micro., 139: 24.
23-2429 (1993))).

Thus, in the case of PCR, AAV nucleic acids are prepared, together with a positive control and a negative control, and subjected to a PCR reaction.
Strategic identification of specific PCR primers allows for the differentiation of wild type from recombinant virus in a rapid and efficient manner. Thus, primers are selected to be specific for wild-type AAV or for wild-type AAV induced through recombination of a helper plasmid and a vector plasmid.

The pSub201 vector (Samulski et al., J. Virol., 61: 3
096-3101 (1987)) or other vectors made to have unique sequences in the left and right arms of the vector, distinguish wild type virus from wild type virus resulting from recombination. To be able to The left and right arms are meant to refer to those portions of the vector that flank the cloning site containing the transgene. Therefore, pSub2
In the case of 01, the left arm is located to the left of the Xbal site and the right arm is located to the right of the Xbal site. These arms contain a hairpin structure.

Referring to the exemplified pSub201 vector to illustrate this method, it is observed that the pSub201 has sequences normally found in the right arm of the AAV on either side of the transgene. Thus, sequences normally found only in the right arm are also present in the left arm, and this native left sequence has been deleted.

Thus, on both sides of the virus, the presence or absence of the left sequence or the presence of the right sequence is produced by recombination between the helper plasmid containing the right sequence and the vector plasmid on both sides of the transgene. Any wild type AA
Primers can be constructed such that they can be used to identify wild-type AAV containing the normally occurring left sequence from V.

For example, when using the pSub201 vector or an equivalent vector containing the right arm sequence in both arms, the primers that hybridize to the sequence in the AAV ITR in the AAV rep gene; in the AAV splice region; and in the AAVcap gene are: Can be diagnostic.

For the primers, once the appropriate sites have two genomes (these are found to be diagnostic indicators of wild-type virus and cross-generated wild-type virus)
It is well understood that the exact nucleotide sequence and length of any one primer can be varied without diminishing the purpose of the diagnostic assay. Limitations on the variation of these primers depend, for example, on the reaction conditions of the PCR to ensure hybridization of the primer to the target.

In the case of NASBA or 3SR, essentially the same primers can be used, which are configured to include a suitable RNA polymerase promoter.
Other primers for the synthesis of double-stranded intermediates include, for example, Cap2 or AA
It may depend on the use of the V2S2 primer.

The invention encompasses pharmacological inhibition in vivo or ex vivo for treating FVIII-related disorders. RAAV packaged in viral particles
The vector, which may include the rAAV vector (including the ITR), is administered to the subject in an amount effective to obtain the desired factor VIII activity in the serum.

Administration can be by any means by which the therapeutic polypeptide is delivered to the desired target cells. For example, both in vivo and ex vivo methods are contemplated. Intravenous injection of the rAAV vector into the portal vein is a suitable method of administration for transducing hepatocytes. Other in vivo methods include, for example, direct injection into the liver lobe or bile duct and direct injection into the distal liver. Ex vivo dosage forms include in vitro transduction of excised hepatocytes or other cells of the liver with the rAAV vector, followed by transduction and excision of the excised hepatocytes in a portal vein or bile tree of a human patient. To be injected back into (for example, Gr
ossman et al., Nature Genetics, 6: 335-341 (19
94)).

Whether transduction of hepatocytes occurred in vivo or ex vivo, rA
AV vector virus particles, alone or partially hepatectomy, helper virus (
For example, adenovirus, including CMV or HSV-1) or with a secondary agent to enhance transduction efficiency.

The effective amount of rAAV vector to be administered will vary from patient to patient. Therefore,
An effective amount is optimally determined by the physician administering the rAAV vector, and appropriate dosages can be readily determined by one skilled in the art. Useful initial amount for administration can range from 10 ⁹ to 10 ²⁰ particles and possibly 10 ^{9-10 15} particles of each vector for 70kg adult. Generally, a minimum effective amount of virus is desired. After expression of the rAAV vector for a sufficient time (eg, typically 4 to 1
5 days), analysis of factor or factor VIII activity at serum or other tissue levels and comparison with initial levels prior to administration determines whether the amount administered is too small, within the appropriate range, or too large. decide.

Suitable regimes for initial administration and subsequent administrations can also vary, but typically the initial administration is followed by administration as needed. Subsequent doses may be administered at various intervals, ranging from daily to once a year to once every several years. One skilled in the art understands that appropriate immunosuppressive techniques can be recommended to avoid the inhibition or blocking of transduction by immunosuppression of the rAAV viral vector (eg, Vilquin et al., Human Gene Ther., 6:13).
91-1401 (1995)).

Formulations for both ex vivo and in vivo administration include suspensions or emulsions in liquids. The active ingredient (rAAV vector) is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, stabilizers or other reagents that enhance the effectiveness of the pharmaceutical composition.

Pharmaceutical compositions can be prepared as injectable formulations for administration, as is known in the art, and include the use of implantable pumps, which are known in the art and For example, described in US Pat. No. 5,474,552). Numerous formulations for oral or parenteral administration are known and can be used in the practice of the present invention. This vector can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

For parenteral administration, solutions in adjuvants such as sesame or peanut oil or in aqueous propylene glycol and sterile aqueous solutions may be employed. Such aqueous solutions may be buffered if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of the AAV vector as a free acid (DNA contains acidic phosphate groups) or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions of AAV virus particles can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, the preparation contains a preservative to prevent the growth of microorganisms. The sterile aqueous medium employed is available by standard techniques well known to those skilled in the art.

Pharmaceutical forms suitable for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form must be sterile and must be fluid to the extent that parenteral administration is possible. The formulation must be stable under the conditions of manufacture and storage and must be able to prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier is a solvent or dispersion medium (these are
For example, it can be water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), including suitable mixtures thereof and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

A sterile parenteral formulation can be obtained by incorporating the AAV vector in the required amount in an appropriate solvent, as appropriate, with the various other ingredients listed above, followed by sterilization, such as filtration. It is prepared by performing Generally, dispersions are prepared by incorporating the sterile active component into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated under a vapor. In the case of sterile powders for the preparation of a sterile injectable solution, the preferred methods of preparation are vacuum drying and lyophilization, which result in a powder of the active ingredient and any further desired ingredients.

[0086] The recombinant vectors of the present invention can also be administered orally. Such preparations may be formulated as is well known in the art, and may be preserved, for example, by including flavors, flavors, coloring and the like.

While the invention has been described generally above, the invention can be better understood by reference to the following examples. The following examples are provided for illustrative purposes only, and are not considered limitations of the present invention.

EXAMPLES AAVMFGSF8-HC is an AAV vector in which the human FVIII heavy chain coding sequence (the subsequently introduced translation stop codon) is under the transcriptional control of MLV LTRs and introns from MFG (Dranoff et al., PNAS, 90: 353
9-3543 (1993)). The polyadenylation sequence may be a bovine growth hormone (bG
H) Derived from the gene.

AAVMFFGSF8-LC is a human FVIII light chain coding sequence that is AAVVM
AAV vector replaced with the human FVIII heavy chain sequence of FGSF8-HC. Both vectors contain the signal sequence for the human FVIII heavy chain.

AAVMGFGSF8-HC was digested with BamHI and NcoI to obtain pSSV
9-MFG-S was converted to MFG-hFVIIIΔB (Dwarki et al., PNAS, 9
2: 1023-1027 (1995)) from 1.8 kb NcoI-Asp7.
0.48 kb Asp718-B from the 18 fragment and pTAF8-HC
It was generated by ligation to the amHI fragment. pTAF8-HC,
The TA cloning vector pCR2.1 (Invitrogen) was converted to MFG-
Using hFVIIIAB as a template, oligonucleotide primer 5
'-AAGCTGGTACCTCACAGAA-3' (SEQ ID NO: 1) and 5 '
-TCGATGGATCCTCATTAATTCTGGGAGAAGCTTTCT
It was obtained by ligation with a fragment generated by PCR using TGG-3 ′ (SEQ ID NO: 2).

AAVMGFGSF8-LC was digested with BamHI and AgeI digested pSSV9
-MFG-S was converted to a 0.26 kb AgeI-ScaI fragment from pTasig, a 0.78 kb HincII-NdeI fragment from pTAF8-LC and a 1.54 kb NdeI-Bam from MFG-hFVIIIΔB.
Produced by ligation to the HI fragment. pTasig (this is F
VIII signal sequence and a ScaI site), a TA cloning vector pCR2.1 (Invitrogen), MFG-hFVIIIΔB as a template, and an oligonucleotide primer 5′-ACAGGCTCTCTA
ACTTAGT-3 '(SEQ ID NO: 3) and 5'-TTAGCAGTACTAAA
It was generated by ligating with a fragment generated by PCR using AGCAGAATCGCAAAA-3 ′ (SEQ ID NO: 4). pTAF8-LC (which contains the FVIII light chain coding sequence preceded by a HincII site)
The cloning vector pCR2.1 (Invitrogen) was converted to MFG-hF
Using VIIIΔB as a template, oligonucleotide primer 5′-GTC
GACCCTCTTGCTTGG-3 ′ (SEQ ID NO: 5) and 5′-AATTC
PCR using CCATATGATGTTGCACTTT-3 ′ (SEQ ID NO: 6)
By ligating the fragments generated by

[0092] The recombinant AAV vector was cloned from Snyder et al., Current Protocol.
Packaging was performed as described in ls in Human Genetics, pages 12.1.1-12.1.24, edited by Dracopol et al., John Wiley & Sons, New York, 1996.

To make the rAAV viral vector, the plasmid is converted to an AAV helper plasmid (which provides the necessary AAV replication and structural proteins,
(Which lacks the AV terminus and therefore cannot be packaged into the virus) were co-transfected into 293 cells via the calcium phosphate method. (Graham
J. et al. Gen. Virol. , 36: 59-74 (1977)) (the cells constitutively express adenovirus E1a protein). A suitable helper plasmid is pDCG2-1 (Li et al., J. Virol., 71: 5236-).
5343 (1997)). The next day, the co-transfected 293 cells were transformed with an adenovirus type 5 strain (eg, adenovirus dl312 strain or d.
1309 strain (Jones & Shenk, Cell, 17: 683-689 (
1979)) to provide the remaining replication and packaging machinery.
After sufficient cytopathic effect, the virus was collected by multiple freeze / thaw cycles and purified according to Snyder et al., (1996), supra. Suitable nucleases for removing unwanted nucleic acids are Benzonas at 250 U / ml.
e (Nycomed, Copenhagen, Denmark). The resulting virus stock consisted of the packaged rAAV vector and progeny helper adenovirus. This helper virus was replaced with cesium chloride (C
(sCl) was removed by purification on a density gradient. The virus stock was then
C. for 30 minutes to inactivate the remaining helper adenovirus (Samu
Iski et al., (1989), supra). Vector titers were obtained by dot blot hybridization (Snyder et al., (1996), supra).

[0094] The culture medium is removed and known methods (eg, ELISA (Connel)
ly et al., Blood, 87: 4671-4677 (1996)) and functional tests (e.g., COATest (KabiVitrum, Stockholm, Sw).
eden))) for human factor VIII activity.

[0095] Immunoreactive factor VIII and factor VIII activity were observed.

[0096] All references mentioned herein are hereby incorporated by reference.

Now that the invention has been fully described, one of ordinary skill in the art will recognize many changes and modifications therefrom.
It is evident that modifications can be made to the invention without departing from the spirit or scope of the appended claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows rAAV-MFG-human factor VIII HC vector and rAAV.
-Schematic diagram of the MFG-human factor VIII LC vector. ITR: AAV inverted terminal repeat; MFG promoter: mouse Moloney virus long terminal repeat; ML
VIVS: mRNA splice donor / splice acceptor of mouse leukocyte virus; huF8HC: human factor VIII heavy chain gene; hu8LC: human factor VIII light chain gene; bGH pA: bovine growth hormone polyadenylation site.

FIG. 2 shows that the functional factor VIII is rAAV-MFG-human factor VIII H
FIG. 6 is a graph showing that each of 1 × 10 ¹⁰ particles of C vector and rAAV-MFG-human factor VIII LC vector is produced after infecting 293 cells in the presence of adenovirus. Factor VIII activity was measured by the COATest and ChromZ assays. Detect only when both vectors are present. GFP is described in Zolotukhin et al. Virol. , 70:
AAV vector expressing the green fluorescent protein described in 4646-4654 (1996), and serves as a negative control.

FIG. 3 is a diagram of the pSSV9-MFG-S vector.

FIG. 4 is a diagram of an AAV-MFGSF8-LC vector.

FIG. 5 is a diagram of an AAV-MFGSF8-HC vector.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Kout, Linda California 94588, Pleasanton , Oak Creek Drive 7814 F term (reference) 4B024 AA01 BA14 CA04 DA03 EA04 GA11 HA17 4C084 AA13 DC15 NA14 ZA531 4C087 AA01 BC83 CA12 NA14 ZA53

Claims (28)

[Claims] 1. A method for obtaining human factor VIII activity in a subject, comprising: providing a cell capable of expressing factor VIII with a plurality of recombinant adeno-associated viruses (rAA).
V) administering a vector, wherein each of the vectors expresses one or more domains of human factor VIII, and wherein the expressed domains do not have a B domain; A method comprising a light chain and a heavy chain of the factor. 2. The method of claim 1, wherein said subject is a mammal. 3. The method of claim 2, wherein the subject's liver cells are transduced ex vivo with the rAAV vector, and wherein the administering delivers the transduced liver cells to the portal vasculature of the mammal. The method of claim 1, comprising: 4. The method of claim 3, wherein said subject is a mammal. 5. The method of claim 1, wherein the first vector expresses a human factor VIII heavy chain and the second vector expresses a human factor VIII light chain. 6. The method of claim 1, wherein the rAAV vector comprises: a promoter capable of expressing human factor VIII; a structural gene encoding one or more domains of human factor VIII. And two AAV inverted terminal repeats flanking the promoter and the structural gene;
Including, methods. 7. The method of claim 6, wherein the inverted terminal repeat comprises a portion of a wild type AAV inverted terminal repeat. 8. The method of claim 6, wherein said promoter is obtained from a virus. 9. The method of claim 8, wherein said virus is a murine leukemia virus. 10. The method of claim 9, wherein said mouse leukemia virus is Moloney murine leukemia virus. 11. The method of claim 6, wherein said rAAV vector further comprises a signal peptide sequence upstream of said structural gene. 12. The method of claim 11, wherein said signal peptide sequence is a human factor VIII signal peptide sequence. 13. The method of claim 1, further comprising providing a partial hepatectomy to the subject. 14. The method of claim 1, further comprising administering a helper virus to the subject's portal vasculature. 15. The method of claim 1, further comprising administering a secondary factor to the liver cells of the subject to enhance transduction efficiency. 16. The method of claim 15, wherein said administration of said secondary factor and said administration of said recombinant AAV vector occur in vivo. 17. The method of claim 3, wherein a secondary factor is applied to said liver cells of said subject to enhance transduction with said recombinant AAV vector ex vivo. 18. The method of claim 1, wherein the administering comprises: injecting the recombinant AAV vector into the subject's portal vasculature. 19. The method of claim 18, wherein said subject is a mammal. 20. The method of claim 1, wherein the factor VIII activity is detectable by an increase in the blood level of the subject as compared to the factor VIII activity in the subject's blood prior to the administering step. The described method. 21. The method of claim 3, wherein said liver cells are hepatocytes. 22. The method of claim 6, wherein the first rAAV vector expresses a human factor VIII heavy chain and the second rAAV vector expresses a human factor VIII light chain. 23. A pharmaceutical composition comprising: a plurality of recombinant adeno-associated (rAAV) vectors, each one of human factor VIII operably linked to a promoter sequence and a polyadenylation sequence. A vector comprising a polynucleotide encoding one or more domains; and a pharmaceutically acceptable carrier, excipient or diluent, wherein the plurality of rAAV vectors comprise a B-domain-free, factor VIII Pharmaceutical compositions that express heavy and light chains. 24. The composition of claim 23, wherein said promoter is obtained from a virus. 25. The virus of claim 24, wherein the virus is a murine leukemia virus.
A composition according to claim 1. 26. The composition of claim 25, wherein said virus is Moloney murine leukemia virus. 27. The composition of claim 23, wherein said recombinant AAV vector further comprises a signal peptide sequence upstream of said polynucleotide encoding one or more domains of human factor VIII. 28. The composition of claim 27, wherein said signal sequence is a human factor VIII signal peptide sequence.