Biochemical Indicators
Biological Dosimetry
Chromosomal morphology
Cloning
Cytotrophoblasts
Deoxyadenosine triphosphate
Deoxycytosine triphosphate
Deoxyguanosine triphosphate
Deoxynucleotide triphosphate
Deoxythymidine triphosphate
Deoxyuridine triphosphate
Disease susceptibility
Down syndrome
Edward syndrome
Environmental Agents
Fetal cells
Fetal trophoblasts
Flow Cytometry
Gene Expression
Gene therapy
Genetic Damage
Genetic counseling
Genetic engineering techniques
Genome Databases
Genome Mapping
Haploid genome
Hybridization
Hybridization Probes
Insert libraries
Karyotype
Maternal blood
Metabolic Pathways
Mitotic metaphase
Molecular turnover
Pathway Analysis
Prenatal Diagnosis
Protein identification
Protein interactions
Protein products
Protein quantitation
Protein resolution
Proteomics
Radiation Injury
Subcellular localizations
Synthetic Oligonucleotides
Translational modifications
Translocations
Trisomy
X Chromosome
bicarbonate buffer
biological dosimetry
cell nucleus
chemical mutagens
chromosomal abnormalities
chromosomal material
clastogenic agents
cytogenetic analyses
cytogenetic research
detection of disease
dicentric chromosomes
ethylenediaminetetraacetate
fluorescein isothiocyanate
fluorescence-activated cell sorting
genetic rearrangements
genetic screening
halogenated hydrocarbons
human chromosomes
in situ hybridization
interphase chromosomes
ionizing radiation
molecular cytogenetics
myelogenous leukemia
nucleic acid probes
phenylmethylsulfonyl fluoride
phytohemagglutinin
propidium iodide
retinoblastoma
sodium dodecyl sulfate
staining patterns
tumor cytogenetics
uterine cancers
About Proteins
So what's happening in the genetic applications frontiers?
Transkaryotic Therapies, Inc located in Cambridge, Massachusetts, is developing production of human proteins and products geared at treatment of rare genetic diseases. Products currently being tested in human clinical trials include Gene-Activated erythropoietin (GA-EPO) for the treatment of anemia, alpha-galactosidase A (alpha-gal) for the treatment of Fabry disease, and Factor VIII for the treatment of hemophilia A. The firm announced completion of Phase II testing of Replagal™ (alpha-galactosidase A) for the treatment of Fabry disease. It is also developing treatments for Hunter syndrome, Gaucher disease, and other rare genetic disorders.
According to the discussion in one of their patents -- "The
ability to move DNA from one cell to another is a powerful tool in modern
molecular biology, yet the idea that this movement might be possible
predates the current revolution in genetic engineering. In 1928, Griffith
paved the way for the discovery that nucleic acids are the genetic
material when he noticed that the virulence of bacteria could be altered
by mixing live bacteria with solutions derived from killed bacteria. By
the early 1960's, not only was the structure of the relevant component of
the solution, DNA, solved, but it was already established that DNA could
be moved into mammalian cells (Syzbalski, 1961). The focus of these early
days of molecular biology and tissue culture were irreversibly changed by
two critical developments: the discovery of calcium phosphate
precipitation, a simple procedure to introduce DNA into immortalized cells
in culture (Graham and van der Eb, 1972) and the isolation and
characterization of mammalian globin, insulin, and growth hormone genes in
the mid- to-late 1970's.
Today, the ability to manipulate DNA and
to introduce it into cells has profound practical implications for human
health. Recombinant proteins produced by such manipulations are becoming
widely accepted treatments for a number of human diseases and play major
roles in agriculture. Though far less developed, the field of human gene
therapy also has been and will continue to be influenced by improvements
in technologies for the manipulation of DNA. "
Information obtained from patent disclosure:
| United States Patent | 6,063,630 |
| Treco , et al. | May 16, 2000 |
Targeted introduction of DNA into primary or
secondary cells and their use for gene therapy
The present invention relates to a method of gene or DNA targeting in cells of vertebrate, particularly mammalian, origin. That is, it relates to a method of introducing DNA into primary or secondary cells of vertebrate origin through homologous recombination or targeting of the DNA, which is introduced into genomic DNA of the primary or secondary cells at a preselected site. The present invention further relates to primary or secondary cells, referred to as homologously recombinant (HR) primary or secondary cells, produced by the present method and to uses of the homologously recombinant primary or secondary cells. The present invention also relates to a method of turning on a gene present in primary cells, secondary cells or immortalized cells of vertebrate origin, which is normally not expressed in the cells or is not expressed at significant levels in the cells.
Isn't this fasinating? - Find out more ... the details of the patent are provided further below, but before getting there ... consider this ...
In order to gain further understanding of proteins and their possibilities we've gathered references relating to proteins, pioinformatics, and other relevant topics as well as the key patents associate with gene activation proteins.
KEY TEXTS
Before continuing with our discussion, ... we must bring this to your attention. If you treasure culture and scholarly work, here's two recent must haves:
Our Posthuman Future by Francis Fukuyama -- discusses cloning, germ-line genetic engineering, stem cell research, neuropharmacology, anti-aging medicine. His basic concern is the potential for violations of human nature that spring from the new biotechnology.
A New Kind of Science by Stephen Wolfram is a different kind of book. It is
written by a practicing master of one of the most intense and prductive
areas of human intellectual and applied endeavor -- Wolfram outlines a
fundamental new way of modeling complex systems
2-D Proteome Analysis Protocols (Methods in Molecular
Biology (Cloth), 112)
by Andrew J. Link(Editor).
Hardcover (January 1999)
Adhesion Protein Protocols (Methods in Molecular
Biology, Vol 96)
by Elisabetta Dejana(Editor), Monica
Corada (Editor). Hardcover (February 1999)
Adhesion Receptors As Therapeutic Targets
by Michael A. Horton(Editor). Hardcover (October 1995)
Advanced Dairy Chemistry : Proteins
by P.
F. Fox(Editor). Hardcover (November 1992)
Details of patent 6,063,630:
BACKGROUND OF THE INVENTION
The ability to move DNA from
one cell to another is a powerful tool in modern molecular biology, yet
the idea that this movement might be possible predates the current
revolution in genetic engineering. In 1928, Griffith paved the way for the
discovery that nucleic acids are the genetic material when he noticed that
the virulence of bacteria could be altered by mixing live bacteria with
solutions derived from killed bacteria. By the early 1960's, not only was
the structure of the relevant component of the solution, DNA, solved, but
it was already established that DNA could be moved into mammalian cells
(Syzbalski, 1961). The focus of these early days of molecular biology and
tissue culture were irreversibly changed by two critical developments: the
discovery of calcium phosphate precipitation, a simple procedure to
introduce DNA into immortalized cells in culture (Graham and van der Eb,
1972) and the isolation and characterization of mammalian globin, insulin,
and growth hormone genes in the mid- to-late 1970's.
Today, the
ability to manipulate DNA and to introduce it into cells has profound
practical implications for human health. Recombinant proteins produced by
such manipulations are becoming widely accepted treatments for a number of
human diseases and play major roles in agriculture. Though far less
developed, the field of human gene therapy also has been and will continue
to be influenced by improvements in technologies for the manipulation of
DNA.
Gene therapy is a medical intervention in which a small
number of the patient's cells are modified genetically to treat or cure
any condition, regardless of etiology, that will be ameliorated by the
long-term delivery of a therapeutic protein. Gene therapy can, therefore,
be thought of as an in vivo protein production and delivery system, and
almost all diseases that are currently treated by the administration of
proteins (as well as several diseases for which no treatment is currently
available), are candidates for treatment using gene therapy. The field can
be divided into two areas: germ cell and somatic cell gene therapy. Germ
cell gene therapy refers to the modification of sperm cells, egg cells,
zygotes or early stage embryos. On the basis of both ethical and practical
criteria, germ cell gene therapy is inappropriate for human use. From an
ethical perspective, modifying the germ line would change not only the
patient, but also the patient's offspring and, to a small but significant
extent, the human gene pool as a whole.
In contrast to germ cell
gene therapy, somatic cell gene therapy would affect only the person under
treatment (somatic cells are cells that are not capable of developing into
whole individuals and include all of the body's cells with the exception
of the germ cells). As such, somatic cell gene therapy is a reasonable
approach to the treatment and cure of certain disorders in human beings.
In a somatic cell gene therapy system, somatic cells (i.e., fibroblasts,
hepatocytes or endothelial cells) are removed from the patient, the cells
are cultured in vitro, the gene(s) of therapeutic interest are added to
the cells and the genetically-engineered cells are characterized and
reintroduced into the patient. The means by which these five steps are
carried out are the distinguishing features of a given gene therapy
system.
To provide an overview of how somatic cell gene therapy
might be applied in practice, an example concerning the treatment of
hemophilia B will be considered. Hemophilia B is a bleeding disorder that
is caused by a deficiency in Factor IX, a protein normally found in the
blood. As a candidate for a gene therapy cure, an affected patient would
have an appropriate tissue removed (i.e., bone marrow biopsy to recover
hematopoietic stem cells, phlebotomy to obtain peripheral leukocytes, a
liver biopsy to obtain hepatocytes or a punch biopsy to obtain fibroblasts
or keratinocytes). The patient's cells would be isolated, genetically
engineered to contain an additional Factor IX gene that directs production
of the missing Factor IX and reintroduced into the patient. The patient is
now capable of producing his or her own Factor IX and is no longer a
Hemophiliac. The physician will most likely schedule close follow up in
the weeks and months after the treatment, but in a literal sense, the
patient would have been cured.
In state-of-the-art somatic cell
gene therapy systems, it is not possible to direct or target the
additional therapeutic DNA to a preselected site in the genome. In fact,
in retrovirus-mediated gene therapy, the most widely utilized experimental
system retroviruses integrate randomly into independent chromosomal sites
in millions to billions of cells. This mixture of infected cells is
problematic in two senses: first, since integration site plays a role in
the function of the therapeutic DNA, each cell has a different level of
function and, second, since the integration of DNA into the genome can
trigger undesired events such as the generation of tumorigenic cells, the
likelihood of such events is dramatically increased when millions to
billions of independent integrations occur.
The problems of
populations consisting of large numbers of independent integrants might be
avoided in two ways. First, a single cell with a random integration site
could be propagated until sufficient numbers of the cloned cell could be
introduced into the individual. The cells that make up this clonal
population would all function identically. In addition, only a single
integration site would be present in the clonal population, significantly
reducing the possibility of a deleterious event. Second, a single cell or
a population of cells could be treated with therapeutic DNA such that the
DNA sequences integrate into a preselected site in the genome. In this
case, all the cells would be engineered identically and function
identically. Furthermore, the risk of a deleterious integration event
would be eliminated. Both the above solutions are demonstrated in this
application.
The application of targeting to somatic cell gene
therapy has several other advantages in addition to simply introducing
additional genes or functional DNA sequences into a cell. In targeted gene
therapy, it would be possible to repair, alter, replace or delete DNA
sequences within the cell. In the illustration of somatic cell gene
therapy discussed above, for example, targeting would allow the patient's
non-functional Factor IX gene to be repaired. The ability to repair,
alter, replace and delete DNA sequences utilizing targeting technology
would expand the range of diseases suitable for treatment using gene
therapy (and for the in vitro production of recombinant proteins as well).
As the above discussion suggests, it would be extremely useful to be able
to target primary and secondary vertebrate cells.
SUMMARY OF THE
INVENTION
The present invention relates to a method of gene or DNA
targeting in cells of vertebrate, particularly mammalian, origin. That is,
it relates to a method of introducing DNA into primary or secondary cells
of vertebrate origin through homologous recombination or targeting of the
DNA, which is introduced into genomic DNA of the primary or secondary
cells at a preselected site. The preselected site determines the targeting
sequences used. The present invention further relates to homologously
recombinant primary or secondary cells, referred to as homologously
recombinant (HR) primary or secondary cells, produced by the present
method and to uses of the HR primary or secondary cells. The present
invention also relates to a method of turning on a gene present in primary
cells, secondary cells or immortalized cells of vertebrate origin, which
is normally not expressed in the cells or is not expressed at significant
levels in the cells. Homologous recombination or targeting is used to
replace the regulatory region normally associated with the gene with a
regulatory sequence which causes the gene to be expressed at significant
levels in the cell.
As described herein, Applicants have
demonstrated gene or DNA targeting in primary and secondary cells of
mammalian origin. Prior to the present work, gene targeting had been
reported only for immortalized tissue culture cell lines (Mansour, Nature
336:348-352 (1988); Shesely, PNAS 88:4294-4298 (1991); Capecchi, M. R.,
Trends in Genetics 5:70-76 (1989)). As a result of the work described
herein, it is now possible to stably integrate exogenous DNA into genomic
DNA of a host or recipient primary or secondary cell. The exogenous DNA
either encodes a product, such as a therapeutic protein or RNA, to be
expressed in primary or secondary cells or is itself a therapeutic product
or other product whose function in primary or secondary cells is desired.
As used herein, the term primary cell includes cells present in a
suspension of cells isolated from a vertebrate tissue source (prior to
their being plated, i.e., attached to a tissue culture substrate such as a
dish or flask), cells present in an explant derived from tissue, both of
the previous types of cells plated for the first time, and cell
suspensions derived from these plated cells. The term secondary cell or
cell strain refers to cells at all subsequent steps in culturing. That is,
the first time a plated primary cell is removed from the culture substrate
and replated (passaged), it is referred to herein as a secondary cell, as
are all cells in subsequent passages. Secondary cells are cell strains
which consist of secondary cells which have been passaged one or more
times. A cell strain consists of secondary cells that: 1) have been
passaged one or more times; 2) exhibit a finite number of mean population
doublings in culture; 3) exhibit the properties of contact-inhibited,
anchorage dependent growth (anchorage-dependence does not apply to cells
that are propagated in suspension culture); and 4) are not immortalized. A
"clonal cell strain" is defined as a cell strain that is derived from a
single founder cell. A "heterogenous cell strain" is defined as a cell
strain that is derived from two or more founder cells.
In the
method of the present invention, cells to be transfected with exogenous
DNA are combined with a DNA construct comprising the exogenous DNA,
targeting DNA sequences and, optionally, DNA encoding one or more
selectable markers and the resulting combination is treated in such a
manner that the DNA construct enters the cells. This is accomplished by
subjecting the combination to electroporation, microinjection, or other
method of introducing DNA into vertebrate cells (e.g., calcium phosphate
precipitation, modified calcium phosphate precipitation, microprojectile
bombardment, fusion methodologies, receptor mediated transfer, or
polybrene precipitation). Once in the cell, the exogenous DNA is
integrated into the cell's genomic DNA by homologous recombination between
DNA sequences in the DNA construct and DNA sequences in the genomic DNA.
The sequences involved in targeting (i.e., those which participate in
homologous recombination with genomic sequences) can be part of the
exogenous DNA or can be separate from (in addition to) the exogenous DNA.
The result is homologously recombinant (HR) primary or secondary cells in
which the exogenous DNA, as well as other DNA sequences present in the DNA
construct, are stably integrated into genomic DNA.
The present
method of targeting exogenous DNA has a wide variety of applications.
These applications fall into three general types or categories: 1)
addition of DNA to sequences already present in vertebrate cells; 2)
replacement of DNA sequences present in vertebrate cells; and 3) deletion
of sequences normally present in vertebrate cells. For example, the
present method can be used to modify primary or secondary cells in order
to repair, alter, delete or replace a resident (host cell) gene; to
introduce a gene encoding a therapeutic or other product not expressed at
significant levels in the primary or secondary cells as obtained; to
introduce regulatory sequences into primary or secondary cells; to repair,
alter, delete or replace regulatory sequences present in primary or
secondary cells; to knock out (inactivate) or remove an entire gene or a
gene portion; to produce universal donor cells (e.g., by knocking out cell
surface antigens), and to augment production of a gene product already
made in the HR primary or secondary cell.
The present method is
particularly useful for producing homologously recombinant cells to be
used for in vivo protein production and delivery, as described in commonly
owned U.S. patent application entitled "In Vivo Protein Production and
Delivery System for Gene Therapy", U.S. Ser. No. 07/787,840, filed of even
date herewith. The teachings of the patent application entitled "In Vivo
Protein Production and Delivery System for Gene Therapy" are incorporated
herein by reference.
The present method of targeting is
particularly useful to turn on a gene which is present in a cell (primary,
secondary or immortalized) but is not expressed in or is not expressed at
significant levels in the cells as obtained. The present method can be
used for protein production in vitro or for gene therapy. For example, it
can be used to turn on genes, such as the human erythropoietin, growth
hormone and insulin genes and other genes (e.g., genes encoding Factor
VIII, Factor IX, erythropoietin, alpha-1 antitrypsin, calcitonin,
glucocerebrosidase, growth hormone, low density lipoprotein (LDL)
receptor, IL-2 receptor and its antagonists, insulin, globin,
immunoglobulins, catalytic antibodies, the interleukins, insulin-like
growth factors, superoxide dismutase, immune response modifiers,
parathyroid hormone, interferons, nerve growth factors, tissue plasminogen
activators, and colony stimulating factors) in a cell of any type
(primary, secondary or immortalized). In this embodiment, a gene's
existing regulatory region can be replaced with a regulatory sequence
(from a different gene or a novel regulatory sequence made by genetic
engineering techniques) whose presence in the cell results in expression
of the gene. Such regulatory sequences may be comprised of promoters,
enhancers, Scaffold-attachment regions, negative regulatory elements,
transcriptional initiation sites, regulatory protein binding sites or
combinations of these sequences. As a result, an endogenous copy of a gene
encoding a desired gene product is turned on (expressed) and an exogenous
copy of the gene need not be introduced.
KEY PATENTS, from the US PATENT DATABASE