Title: Method of stable integration of DNA into neurons
United States Patent: 6,555,369
Issued: April 29, 2003
Inventors: Neuman; Toomas (Santa Monica, CA); Nornes; Howard
O. (Fort Collins, CO)
Assignee: Spinal Cord Society (Fergus Falls, MN)
Appl. No.: 886512
Filed: June 21, 2001
The invention relates to the stable transfection of neurons with DNA
encoding proliferating cell nuclear antigen (PCNA) and replication factor C
(RFC). Also, co-transfection of a functional gene along with the DNA
encoding PCNA and RFC causes stable integration of the functional gene.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present inventors demonstrated that introduced DNA expression was
stable for weeks. With stable integration of the delivered gene into the
genome of the target neurons, this method of gene transfer provides for gene
therapy for neurodegenerative diseases such as Parkinson's, Huntingtons and
Alzheimers and for reconstruction following trauma and stroke. Further, this
method can be used for gene transfer to any non proliferating and fully
differentiated cell of the body, since regulation of the DNA repair and
recombination is not unique in the neuronal cells and, likely, induction of
DNA repair and recombination will result in stable integration of
According to certain embodiments, the invention involves transfection and
expression of DNA repair and recombination factors (for example, PCNA and
large subunit of RFC) that stimulate DNA repair and recombination in
differentiated target cells, in combination with UV irradiation to induce
DNA repair and recombination. There are many factors that stimulate DNA
repair and recombination that may be used in the present invention.
Since both UV and gamma ray irradiation stimulate DNA repair and
recombination processes then it will be possible to use X-ray irradiation
instead of UV irradiation in the present invention. According to certain
embodiments, dosages of X-ray irradiation for in vitro use, may include
exposing cell culture to a cesium (137 Cs) irradiator at doses of about
10 to about 900 rad (1 rad equals 0.01 Gy). Humans and animals may be X-ray
irradiated using an X-ray generator and different dose levels from 0.2 Gy to
50 Gy. According to certain preferred embodiments, irradiation will be
focused on the treated area. Human and animal dosages may be calculated on
results of in vitro experiments and by in vivo experiments as well.
According to certain embodiments, X-rays or other UV irradiation may not be
necessary. Moreover, other means of damaging DNA to induce DNA repair and
reconstruction may be employed according to certain embodiments.
Foreign DNA encoding therapeutically relevant proteins may be cotransfected
during the induced DNA repair and recombination phase, which accomplishes a
stable and functional integration of genes into neurons of different
organisms including humans. This method provides gene-based therapies of the
central nervous system (CNS).
For example, for Parkinson's disease, cDNA encoding tyrosine hydroxylase,
which is key enzyme in the synthesis of L-DOPA and dopamine, can be
introduced into cathecholamine producing neurons. For other
neurodegenerative diseases and for stroke and trauma patients, genes
expressing neurotrophic factors and their receptors can be introduced to
The following example illustrates the invention. This example is for
illustrative purposes only and is not intended to limit the scope of the
Integration of Introduced DNA into Cortical Neurons after Stimulation of DNA
Repair and Recombination Process in Vitro.
The procedure described in Example 1 uses proteins that are known to induce
DNA repair and recombination in postmitotic neurons to achieve stable
integration of DNA was tested using mouse cortical neurons. Cerebral
cortexes of 17 day old mice embryos were dissociated into single cells after
incubation in 0.25% trypsin, 1 mM EDTA for 15 minutes at 37oC.
Trypsin digestion was stopped by DMEM plus 10% fetal calf serum containing
0.1% DNase. Cells (3-4 x105 /ml) were cultured on poly-L-lysine
(5 .mu.g/cm2) and collagen (100 .mu.g/ml) coated 4 chamber culture
slides (LAB-TEK) in Neurobasal medium (GIBCO) containing B27 supplement (GIBCO).
Cytidine arabinoside (10 .mu.M) was present in culture media during days 2-4
to block proliferation of non-neuronal cells. After 8 days in culture, cells
were transfected using LIPOFECTAMINE.TM. reagent according to the
manufacturer's protocol (GIBCO BRL LIFE TECHNOLOGIES) using 5 .mu.g of
pRcCMVneo eukaryotic expression vector (INVITROGEN), IacZ cDNA cloned into
pRcCMVneo (pRcCMV-IacZ), E2F1 cDNA cloned into pRcCMVneo (pRcCMV-E2F1),
E1A135 cDNA cloned into pRcCMVneo (pRcCMV-EIA135), PCNA cDNA
cloned into pRcCMVneo (pRcCMV-PCNA), or RFC cDNA cloned into pRcCMVneo (pRcCMV-RFC)
cDNAs in 1 ml of the LIPOFECTAMINE.TM.. PCNA cDNA was obtained from Dr.
Williams and it has Gene Bank Accession No. X57800. RFC large subunit cDNA
was obtained from Dr. Luckow and it is described, including the nucleic acid
sequence information, in Luckow et al., Molecular and Cellular Biology,
14:1626-1634 (1994). E2F1 cDNA was obtained from Dr. Helm and it has Gene
Bank Accession No. M96577. E1A135 was obtained from Dr. Morain. IacZ
cDNA was obtained from Dr. Gruss.
The cells were exposed to the DNA for 3-5 hours in Neurobasal media and then
placed in neurobasal growth media (GIBCO). Twelve hours after transfection,
the cells were irradiated with a General Electric G8T5 germicidal lamp
emitting predominantly 254-nm light at an incident rate of 0.35 J/m2
/s. After irradiation, fresh neurobasal culture media (GIBCO) containing B27
supplement was added.
Cortical neurons isolated from embryonic day 17 mice embryos differentiate
and maintain a differentiated state in vitro. After 8 days in culture no DNA
synthesis was detected in cortical neurons based on 5-bromo-2'-deoxyundine (BrdU)
incorporation. To determine if the DNA delivered into differentiated
cortical neurons is stably integrated and functional, the pRcCMV-lacZ
discussed above was cotransfected with the pRcCMV-E2F1, pRcCMV-E1A135,
pRcCMV-PCNA, and pRcCMV-RFC discussed above. Transfected cells were analyzed
3 days and 3 weeks after transfection for lacZ expression using X-gal
staining. Three days after transfection, all treatment groups showed very
similar number of lacZ positive cells, which is an indication that
transfection efficiencies were similar in all cultures (close to 1%). After
three weeks, control cultures did not contain any lacZ expressing cells.
Over-expression of PCNA and RFC large subunit, in combination with UV
treatment, results in stable integration of introduced DNA into cortical
neurons in 10% of positively transfected cells. These results also
demonstrated that efficiency of DNA integration into the genome of neurons
is almost 10 times higher after stimulation of DNA repair and recombination
processes compared to induction of S phase of the cell cycle by E1A and E2F
Integration of lacZ into the genome of neurons using long term
cultures of cortical neurons
number of lacZ positive cells
treatment after 3 days after 3 weeks
none 3218 .+-. 203 0
E1A + E2F1 3756 .+-. 312 31 .+-. 7
PCNA + RFC 3257 .+-. 297 103 .+-. 11
PCNA + RFC + UV irradiation 3196 .+-. 301 327 .+-. 29
UV irradiation 3154 .+-. 342 5 .+-. 3
The present inventors also analyzed DNA integration using polymerase chain
reaction (PCR) and Southern blot of genomic DNA of transfected neurons.
Quantitation of PCR and Southern blot analyses using Phosphorimager
technology demonstrated that PCNA+RFC+UV treated cells contain almost 10
times more lacZ DNA than E1A+E2F1 treated cells, and no lacZ DNA was
detected in control cells.
Use of Proteins Known to Stimulate DNA Repair and Recombination and
Integration of DNA into Adult Neurons in Vivo.
Cortical neurons will be transfected with plasmids expressing PCNA and RFC.
The net result of this double transfection will be integration of
cointroduced DNA into genome of neurons.
Adult rats (over 6 weeks) will be anesthetized using ketamine (85 mg/kg) and
xylazine (13 mg/kg). Stereotaxic surgery will be performed to inject 10 .mu.g
of pRcCMV-PCNA/pRcCMV-RFC/cDNA which will be integrated in the same vector
mixture (1:1:1) into the parietal cortex of adult rats. Injections will be
made 4 mm posterior to the bregma, 5-5.5 mm lateral to the midline, and
3.0-3.5 mm depth in the parietal cortex over a five minute period and the
needle will remain in place for an additional 10 minutes. Immunoliposomes
will be prepared as described elsewhere in WO 95/16774, published Jun. 22,
1995, which is incorporated by reference herein. Liposomes will be diluted
to a concentration of 1 mg/ml total lipid. Plasmid DNA and Thy 1.1 antibody
concentrations will be 0.025 mg/ml and 0.25 mg/ml, respectively. Cerebral
cortexes of animals will be X-ray irradiated 1-3 days after surgery using
dose levels from 0.2 Gy to 50 Gy.
Transfected brains will be analyzed 3 days, 7 days, 3 weeks, 2 months, and 6
months after transfection for .beta.-galactosidase expression using X-gal
staining at pH higher than 7.5. This staining minimizes visualization of
endogenous galactosidases and stains the transfected .beta.-gal cDNA will be
transfected alone, without PCNA and RFC cDNAs.
Integration of Tyrosine Hydroxylase (TH) cDNA into Postmitotic Neurons or
Glia in Vivo in Treatment of Parkinson's Disease
Parkinsonism is a slowly progressive neurodegenerative disease of the
central nervous system. Clinical symptoms are tremors at rest, rigidity,
akinesia and postural impairment. A hallmark of the disease is reduction of
the neurotransmitter dopamine in the basal ganglia which is caused by the
loss of nerve cells in the brain stem. These dopamine producing neurons are
located in the substantia nigra nucleus of the mesencephalon and project to
and terminate in the basal ganglia. Major clinical signs and symptoms arise
when around 80% of these neurons are lost.
The administration of the amino acid L-3,4-hydroxyphenylalanine (L-DOPA) is
currently the most common treatment of the disease. L-DOPA is the immediate
precursor of dopamine and after entering the neuron is converted to
dopamine. Remission following this treatment indicates that the remaining
dopamine neurons are adequately adaptive to restore basal ganglia activity.
However, long term systemic L-DOPA treatments are complicated by side
Amelioration of parkinsonian-like deficits in experimental animal models has
also been accomplished by transplantation of fetal dopamine producing cells
into the basal ganglia. With the potential ethical, legal, and
histocompatibility issues associated with the use of fetal cells,
investigators tested the feasibility of using DOPA-secreting cells (chromaffin
cells) dissected from the adrenal medulla. Animal experiments in rodents and
non-human primates using cells from the adrenal medulla, however, have not
been promising because of low survival and immunological rejection (Freed et
al., J Neurosurg., 65:664-670 (1986); Hansen et al., Exp. Neurol., 102:65-75
(1988)). The initial clinical trials with human Parkinson's disease patients
also indicate a need for further basic research (Lindvall et al., Ann. Neuro.,
22:457468 (1987; Goetz et al., N. Engl. J. Med., 320:337-341 (1989)). The
rate-limiting enzyme tyrosine hydroxylase (TH) is involved in the production
of L-DOPA in neurons. The present inventors hypothesize that by increasing
levels of TH at the neurons, one can also obtain L-DOPA at the neurons and
thus treat Parkinson's disease.
This prophetic example is to stably insert a cDNA which codes for the TH
protein into the remaining substantia nigra neurons and in neurons in close
proximity to the substantia nigra neurons in the brain stem of patients with
Parkinson's disease. The advantage of this gene therapy application is that
the TH levels will be elevated in the remaining dopamine neurons of the
substantia nigra and in neurons in close proximity to the substantia nigra
The L-DOPA drug treatments in Parkinson's disease patients have already
demonstrated that the remaining dopamine neurons are capable of restoring
basal ganglia activity. Instead of elevating the neurotransmitter levels in
all the catecholamine/dopamine related pathways which occurs following
systemic L-DOPA treatment, this application will elevate the TH levels only
in the substantia nigra neurons and in neurons in close proximity to the
substantia nigra neurons. The TH levels will be elevated in the specific
dopamine producing neurons, which project to and terminate in basal ganglia.
As discussed above, a hallmark of Parkinson's disease is reduction of the
neurotransmitter dopamine in the basal ganglia.
Immunoliposomes specific for neurons will be made similar to the
immunoliposomes of Example 2 except that TH cDNA is substituted for beta-galactosidase
cDNA. Moreover, one could design a liposome that is specific for a surface
marker of substantia nigra neurons, and thus have a liposome that is even
more specific for those particular neurons. Preferably, the liposomes will
contain about 10-100 .mu.g of the plasmid DNA (pRcCMV-TH, pRcCMV-PCNA, and
pRcCMV-RFC in a 1:1:1 ratio).
Stereotaxic surgery similar to that performed in Example 2 will be performed
to inject liposomes containing the inserted plasmids discussed above locally
into the area of the substantia nigra neurons of a human or other animal. By
selecting the specific area for the injection, one can limit transfection to
the substantia nigra neurons and neurons in close proximity to the
substantia nigra neurons.
Injecting small volumes of cells into brains of human patients is a rather
non-invasive surgery (Lindvall et al. 1987), so injections of liposomes
should not be invasive. One skilled in the art will be able to monitor the
clinical signs of the patient over time for determine the effective dose and
to determine whether subsequent administrations should be provided.
There are additions or alternatives to the above treatment. Given the fact
that the Parkinson-like symptoms can be ameliorated in experimental animal
models by transplanting dopamine producing cells into cells within the basal
ganglia, either glial cells or interneurons in the basal ganglia could be
transfected with the similar cDNA constructs and liposome delivery system.
One skilled in the art would be aware that the targeted liposomes would be
constructed such that they recognize the particular cell type that is to be
Stable Integration of Nerve Growth Factor (NGF) cDNA into Postmitotic Basal
Forebrain Cholinergic Neurons in Alzheimer's Patients
Alzheimer's disease is a progressive neurodegenerative disease of the
central nervous system resulting in senile dementia. Neuronal populations
are differentially affected by the degenerative process with lesions
throughout the brain. The entorhinal cortex and hippocampus are severely
affected and forebrain cholinergic neurons and brain stem serotinergic and
adrenergic neurons which project to the cortex and hippocampus are
particularly vulnerable. There are a variety of cellular pathologies
including the severally affected cytoskeleton (neurofibrillar tangle) and
extracellular deposits of beta-amyloid protein (senile plaques).
It has been proposed that Alzheimers patients be treated with pluripotent
neurotrophic factors (Terry, "Regeneration in Alzheimer Disease and Aging,"
Advances in Neurology, Vol.59, pp. 1-4, Ed. F. J. Seil. Raven Press, Ltd.,
New York (1993)). There is a family of proteins called neurotrophic factors
that have been shown to be responsible for growth and survival of neurons
during development (Levi-Montalcine, Science, 237:1154-1162 (1987); Hofer et
al., Nature, 331:261-261 (1988)) and to prevent death of neurons induced by
lesions (Yan et al., Nature, 360:753-755 (1992; Koliatosos et al., Neuron,
10:359-367 (1993)). In the nervous system, the neurotrophic factors are
synthesized and released from other neurons or support cells (glia). These
factors bind to specific receptors on neurons, resulting in the activation
of metabolic pathways which in turn are responsible for activating the
production of proteins involved with growth and survival.
One of the characteristics of the Alzheimer brain is the reduction of
cortical acetylcholine which can be caused by atrophy and depletion of
nerve-growth-factor dependent (NGF) cholinergic forebrain neurons that
project to the cerebral cortex and hippocampus. In animal models, lesion of
this cholinergic pathway to the hippocampus results in cell loss in the
forebrain cholinergic neurons which can be reversed by NGF (Tuszynski et
al., Ann. Neurol., 30:625-636 (1991)). Recombinant human nerve growth factor
was infused into the lesion site of the adult primate brain.
In this prophetic example, cDNA encoding NGF protein will be stably inserted
into the brain of Alzheimer patients. The source of NGF is Gene Bank
Accession No. V01511. The cDNA would be stably inserted into the forebrain
where damaged cholinergic neurons are localized. The cDNA constructs using
the CMV promoter plasmids and the liposome delivery methods for delivery of
the cDNA to the forebrain neurons would be similar to that described in the
previous examples above. (NGF cDNA, of course, would be substituted for the
TH cDNA.) Moreover, as discussed in Example 3, one skilled in the art would
be able to monitor the patient to determine proper dosages and
There is an addition or alternative to this treatment of Alzheimers
patients. Since the brain derived neurotrophic factor (BDNF) is at low
levels in the hippocampus of Alzheimers patients (Phillips et al., Neuron,
7:695-702 (1990)), and since BDNF promotes survival of forebrain cholinergic
neurons in vitro (Alderson et al., Neuron, 5:297-306 (1990)), cDNA
constructs coding for BDNF protein could also be used to transfect
hippocampal neurons in Alzheimers patients. The cDNA constructs using the
CMV promoter plasmids and the liposome delivery methods for delivery of the
cDNA to the hippocampal neurons would be similar to that described in
Example 3 above.
Claim 1 of 13 Claims
What is claimed is:
1. A set of vectors for inducing a differentiated cell to express introduced
DNA encoding a desired protein, the set of vectors comprising:
DNA encoding a desired protein, nucleic acid encoding proliferating cell
nuclear antigen (PCNA), and nucleic acid encoding replication factor C (RFC).
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