Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-19
2003-05-20
Horlick, Kenneth R. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S069700, C435S325000, C530S350000
Reexamination Certificate
active
06566073
ABSTRACT:
Background of the Invention
Summary of the Invention
Brief Description of the Figures
Detailed Description
Definitions:
Capable of selectively hybridizing
Cells”, “host cells” or “recombinant host cells
Cell line
Composite”, “fusion”, and “recombinant
coding sequence
construct
Derived from
Domain
Expression control element
Gene
Genetically engineered cells
Heterologous
Interact
Nucleic acid
polylinker
Protein”, “polypeptide” and “peptide
recombinant virus
secretory machinery
Transfection
Infection
Transduction
Transgene
vector
Conditional Retention Domains:
Cleavage Enzymes:
Secretory Signal Sequences:
Target proteins:
Disposal Targeting Sequences:
Design and assembly of the DNA constructs
Promoters
Introduction of Constructs into Cells
Introduction of Constructs into Animals
Viral Vectors:
Adenoviral vectors
AAV Vectors
Hybrid Adenovirus-AAV Vectors
Retroviruses
Other Viral Systems
Administration of Viral Vectors
Uses
Methods for identifying CRDs
Pharmaceutical Compositions & Their Administration to Subjects Containing
Engineered Cells
Administration
Compositions
Examples
Example 1: Generation of domains and vectors used for expression of F(36M) fusion proteins
Example 2: Identification and synthesis of a ligand for the conditional retention domain F36M FKBP
Example 3: The conditional retention domain F(36M) FKBP; studies with hGH
Example 4: Localization/cleavage of fusion protein
Example 5: Dose response and kinetics of hGH secretion
Example 6: Regulated insulin secretion
Example 7: Regulated expression of a membrane tethered protein
Example 8: Construction and testing of a construct for conditional secretion of hGH using rat retinol binding protein as a CRD
Example 9: Physiological effects of regulated insulin secretion in vivo
Claims
Abstract
BACKGROUND OF THE INVENTION
A number of important applications, including for example, gene therapy, production of biological materials and materials and methods for biological research, depend on the ability to induce cells to produce proteins of therapeutic, commercial, or experimental value. A variety of regulatable expression systems have been developed, including systems involving allostery-based switches triggered by tetracycline, RU486 or ecdysone, as well as dimerization-based switches triggered by dimerizing agents such as rapamycin, coumermycin, dimers of FK506, synthetic FKBP-binders and/or CsA, or analogs thereof. See e.g. Clackson, “Controlling mammalian gene expression with small molecules” Current Opinion in Chemical Biology, 1:210-218, 1997. In these expression systems, protein production is regulated at the transcriptional level. An inherent limitation of all such systems is the inability to achieve fine temporal control over secretion of the target protein. For example, secretion of maximal, therapeutic levels of the protein is delayed by many hours or even days until the transcribed mRNA accumulates to levels high enough to produce significant amounts of secreted protein. Likewise, secretion cannot return to low baseline levels following removal of the inducing drug until the mRNA is completely degraded, which may also take many hours or days. For many applications this level of control is not sufficient; in these instances, it would be desirable to induce protein production on a much more rapid time scale than that achievable using transcription-based methods.
SUMMARY OF THE INVENTION
This invention takes a unique approach to the regulated production of a target protein, based not on regulated transcription, but on regulated release or secretion of the target protein. Compositions and methods of this invention are useful in biological research and in gene therapy applications.
Key features of the invention include conditional retention domains (“CRDs”), fusion proteins containing them, ligands which bind to the CRDs and permit release or secretion of the fusion proteins, recombinant nucleic acids encoding such fusion proteins, vectors containing such recombinant nucleic acids, cells transduced with these vectors and other materials and important methods involving such. Key fusion proteins of the invention contain at least two mutually heterologous domains, one of which being a CRD.
More specifically, the fusion proteins of this invention are designed to contain at least one conditional retention domain (CRD) and at least one additional domain that is heterologous thereto, usually with a secretory signal sequence. Proteins containing a secretory signal sequence are translated in the endoplasmic reticulum (ER) and then pass through other secretory compartments such as the cis, medial and trans Golgi on their way to being secreted. However, proteins containing one or more CRDs are, as a rule, retained in the secretory machinery except in the presence of a ligand which binds to the protein. Illustrative examples of CRDs include retinol binding proteins and human FKBP 12 mutants such as F36M hFKBP12, as are discussed in detail below. Concatenation of multiple CRDs may allow the user to modulate the degree of aggregation or retention.
Typically the fusion protein also contains a secretory signal sequence to target the fusion protein to a secretory compartment such as the ER or any part of the Golgi apparatus. Many secretory signal sequences are known. Human growth hormone, for example, is the source of a secretory signal sequence suitable for use in this invention.
Additionally, it is preferred in many embodiments that the fusion protein further contain an enzymatic cleavage site such that a portion of the fusion protein containing the CRD can be cleaved from a portion of the fusion protein containing a peptide sequence heterologous to the CRD. Preferably the enzymatic cleavage site comprises a peptide sequence recognized by a trans-Golgi specific endoprotease such as furin. For instance, a cleavage site for furin is provided by the peptide sequence SARNRQKR (SEQ ID NO. 1).
The portion of the fusion protein which is heterologous to the CRD may comprise any protein or protein domain of interest to the practitioner. For instance, the heterologous portion may comprise a target protein such as insulin, parathyroid hormone or beta-endorphin.
To illustrate this further, one typical fusion protein of the invention comprises a signal sequence, a conditional retention domain, a furin cleavage site, and a polypeptide sequence comprising a selected target protein sequence. An example of such a fusion protein comprises, in N-terminal to C-terminal order, a signal sequence from human growth hormone, three F36M hFKBP 12 domains, a human stromelysin-3 furin cleavage site, and a selected target protein sequence. Fusion proteins may also contain several target proteins each separated by an enzymatic cleavage site. For example, such a fusion protein might contain a signal sequence from human growth hormone, one or more copies of a CRD such as F36M hFKBP 12, a furin cleavage site, a target protein, another furin cleavage site and another target protein. This type of construct allows for simultaneous release of more than one target protein.
In addition, the fusion proteins of this invention may optionally comprise a lysosomal targeting signal or other polypeptide sequence targeting it for degradation. By locating such a peptide sequence together with the CRD(s) on one side of the cleavage site and the selected target polypeptide on the other side of the cleavage site, one can help assure cellular removal of the CRD-containing portion of the fusion protein.
One object of the invention is thus the fusion proteins described herein.
Another object of the invention is the recombinant nucleic acids encoding such fusion proteins. Those recombinant nucleic acids may be operably linked to an expression control sequence permitting their expression in host cells into which they have been transduced, or which otherwise contain them. Any promoter may be used to drive expression of these fusion proteins, including strong promoters like the CMV enhancer, other viral promoters such as the RSV promoter or tissue specific promoters like the MCK enhancer.
An
Clackson Timothy
Rivera Victor
Rothman James
Ariad Gene Therapeutics, Inc.
Berstein David L.
Horlick Kenneth R.
Wilder Cynthia
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