DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 76-77, 79-129, 131-133, and 136-152, amended, previously presented, or newly added, are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
While determining whether a specification is enabling, one considers whether the claimed invention provides sufficient guidance to make and use the claimed invention, if not, whether an artisan would require undue experimentation to make and use the claimed invention and whether working examples have been provided. When determining whether a specification meets the enablement requirements, some of the factors that need to be analyzed are: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and whether the quantity of any necessary experimentation to make and use the invention based on the content of the disclosure is “undue”.
Nature of Invention: The claimed invention is directed to pluripotent stem cell (PSC) derived Fox2a+/+LMX1 cell therapy for treating parkinsonism disease in a mammal.
Breadth of the Claims: The claims are drawn to a method of treating a parkinsonism disease in a mammalian subject. The breadth of the therapeutic method encompasses treating any and/or all symptoms any form of parkinsonism in any mammalian subject, including humans. The specification states, “term “parkinsonism” refers to a group of diseases that are all linked to an insufficiency of dopamine in the basal ganglia which is a part of the brain that controls movement. Symptoms include tremor, bradykinesia (extreme slowness of movement), flexed posture, postural instability, and rigidity. A diagnosis of parkinsonism requires the presence of at least two of these symptoms, one of which must be tremor or bradykinesia. The most common form of parkinsonism is idiopathic, or classic, Parkinson's disease (PD), but for a significant minority of diagnoses, about 15 percent of the total, one of the Parkinson's plus syndromes (PPS) may be present. These syndromes also known as atypical parkinsonism, include corticobasal degeneration, Lewy body dementia, multiple systematrophy, and progressive supranuclear palsy. In general, Parkinson's disease involves the malfunction and death of vital nerve cells in the brain primarily in an area of the brain called the substantia nigra. Many of these vital nerve cells make dopamine. When these neurons die off, the amount of dopamine decreases, leaving a person unable to control movement normally. The intestines also have dopamine cells that degenerate in Parkinson's disease patients, and this may be an important causative factor in the gastrointestinal symptoms that are part of the disease. The particular symptoms that an individual experiences can vary from person to person. Primary motor signs of Parkinson's disease include the following: tremor of the hands, arms, legs, jaw and face, bradykinesia or slowness of movement, rigidity or stiffness of the limbs and trunk and postural instability or impaired balance and coordination.” See paragraph [0132]. Thus, the breadth of the claims encompass reducing or alleviating at one symptom of parkinsonism to leaving all symptoms of parkinsonism. As discussed in the specification, parkinsonism is associated with diverge group of primary and secondary symptoms including reducing alleviating neuronal cell death, improving motor movement, reducing/alleviating rigidity and slowness/impaired movement, reducing/alleviating tremors, reducing/alleviating impair gastrointestinal function, among many other symptoms.
The body of the method comprises a fairly narrow method of producing a differentiated cell population of FOX2A and LMX1 positive cells from human pluripotent stem cells and administering an effective amount of the differentiated cell population to the brain of the subject. The claims do not specify how long or when the pluripotent cells are cultured with the listed factors of (a)-(c). As such, the culture can take place for any length of time. Given the amount of time and type of factor exposure results in cells of different maturity (See examples 1 and 2 of the specification for instance). The breadth the cells that are produced from the differentiation methods of step 1, include but are not limited to cells that are undifferentiated cells, non-neural progenitor cell, neural progenitor cells, immature neural cells, mature neural cells, highly differentiated dopaminergic neurons, or a combination thereof. As such, the breadth of the cells that are then administered to the subject is broad with diverse structural/functional elements. The claims does not recite any type of selection procedure or enrichment procedure, as such the breadth of the claims encompasses administering the resultant cells from step (1) directly into subject, wherein the resultant cells is a heterogenous population of cells comprising the any one or all of the above described cell types. The administering step (2) specifies that a therapeutically effective amount of cells is injected/transplanted into the brain. The claim does not specify a region of the brain. As such, the breadth of the administration encompasses injected/transplanting the cells into any part of the brain including those having pathology, those that don’t have pathology, areas of the brain not normally impacted in parkinsonism and areas of the brain that are not normally impacted with parkinsonism. The claims do not specify that the resultant cells comprise no tumorigenic cells and that the method does not result in tumor formation associated with pluripotent cell derivation. Thus the breadth of the claimed method allows for the presence of cells that are tumorigenic and allows for tumor formation to occur.
Specification Guidance: The specification provides the following guidance (citations from pre-grant publication):
[0011] Yet another aspect of the present invention relates to a method of treating a disease in a mammalian subject comprising administering to the subject a therapeutically effective amount of the culture of the present invention or as described above. The mammalian subject may be a human. The disease may be a disease of the central nervous system (CNS). In some embodiments, the disease is Parkinson's disease (PD) or a Parkinson-plus syndrome (PPS). The culture may comprise dopaminergic neurons that are not fully differentiated or are at day 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of differentiation (e.g., day 17-24 of differentiation). In some embodiments, the culture comprises a hyaluronic acid matrix. As shown in the examples, advantages have been observed for administering cells that are at an intermediate stage of differentiation (e.g., dopaminergic neurons that are not fully differentiated) are administered to a mammalian subject.
[0129] B. Treatment of Diseases of the Central Nervous System
[0130] 1. Disease of the Central Nervous System
[0131] Dopaminergic neurons, such as post-mitotic midbrain DA neurons, can be transplanted to regenerate neural cells in an individual having a disease of the central nervous system (CNS). In some embodiments, midbrain DA neurons produced according to methods of the present invention may be administered to a subject to treat a CNS disease (e.g., administered to the brain or midbrain, such as the caudate nucleus, putamen, or substantia nigra to treat Parkinson's Disease). Such diseases can include, but are not limited to, neurodegenerative diseases, such as parkinsonism.
[0132] As used herein, term "parkinsonism" refers to a group of diseases that are all linked to an insufficiency of dopamine in the basal ganglia which is a part of the brain that controls movement. Symptoms include tremor, bradykinesia (extreme slowness of movement), flexed posture, postural instability, and rigidity. A diagnosis of parkinsonism requires the presence of at least two of these symptoms, one of which must be tremor or bradykinesia. The most common form of parkinsonism is idiopathic, or classic, Parkinson's disease (PD), but for a significant minority of diagnoses, about 15 percent of the total, one of the Parkinson's plus syndromes (PPS) may be present. These syndromes also known as atypical parkinsonism, include corticobasal degeneration, Lewy body dementia, multiple systematrophy, and progressive supranuclear palsy. In general, Parkinson's disease involves the malfunction and death of vital nerve cells in the brain primarily in an area of the brain called the substantia nigra. Many of these vital nerve cells make dopamine. When these neurons die off, the amount of dopamine decreases, leaving a person unable to control movement normally. The intestines also have dopamine cells that degenerate in Parkinson's disease patients, and this may be an important causative factor in the gastrointestinal symptoms that are part of the disease. The particular symptoms that an individual experiences can vary from person to person. Primary motor signs of Parkinson's disease include the following: tremor of the hands, arms, legs, jaw and face, bradykinesia or slowness of movement, rigidity or stiffness of the limbs and trunk and postural instability or impaired balance and coordination.
[0133] 2. Methods for Administering Cells
[0134] Stem cells or differentiated cells can be administered to a subject either locally or systemically. Methods for administering DA neurons to a subject are known in the art. If the patient is receiving cells derived from his or her own cells, this is called an autologous transplant; such a transplant has little likelihood of rejection.
[0135] Exemplary methods of administering stem cells or differentiated neuronal cells to a subject, particularly a human subject, include injection or transplantation of the cells into target sites (e.g., striatum and/or substantia nigra) in the subject. The stem cells and/or DA neurons can be inserted into a delivery device which facilitates introduction, by injection or transplantation, of the cells into the subject. Such delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject. In a preferred embodiment, the tubes additionally have a needle, e.g., a syringe, through which the cells of the invention can be introduced into the subject at a desired location. The stem cells can be inserted into such a delivery device, e.g., a syringe, in different forms. For example, the cells can be suspended in a solution, be in cell aggregates, or alternatively embedded in a support matrix when contained in such a delivery device.
[0136] Support matrices in which the stem cells or neurons can be incorporated or embedded include matrices that are recipient-compatible and that degrade into products that are not harmful to the recipient. The support matrices can be natural (e.g., hyaluronic acid, collagen, etc.) and/or synthetic biodegradable matrices. Synthetic biodegradable matrices that may be used include synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid. In some embodiments, dopaminergic neurons (e.g., dopaminergic neurons that are not fully differentiated) are embedded in hyaluronic acid matrix and administered to a subject to treat a neurodegenerative disease (e.g., Parkinson's disease).
[0137] As used herein, the term "solution" includes a pharmaceutically acceptable carrier or diluent in which the cells of the invention remain viable. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is known in the art. The solution is preferably sterile and fluid to the extent that easy syringability exists.
[0138] Preferably, the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. In some embodiments a solution containing DA neurons or midbrain DA neurons is administered to a patient in sterile solution of BSS PLUS (Alcon, Fort Worth, Tex.). If desired a preservative or antibiotic may be included in the pharmaceutical composition for administration. Solutions of the invention can be prepared by incorporating neurons as described herein in a pharmaceutically acceptable carrier or diluent and, other ingredients if desired.
[0139] 3. Dosage and Administration
[0140] In one aspect, the methods described herein provide a method for enhancing engraftment of progenitor cells or DA neurons in a subject. In one embodiment, the subject can be a mammal. In another embodiment, the mammal can be a human, although the invention is effective with respect to all mammals.
[0141] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of each active ingredient required to be administered depend on the judgment of the practitioner and are particular to each individual. However, suitable dosage ranges depend on the route of administration. Suitable regimes for administration are also variable.
[0142] 4. Efficacy
[0143] The efficacy of a given treatment to enhance DA neuron engraftment can be determined by the skilled artisan. However, a treatment is considered "effective treatment," as the term is used herein, if any one or all of the signs or symptoms of e.g., poor DA neuron engraftment are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, e.g., by at least 10% following treatment with a cell population as described herein. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, need for medical interventions (i.e., progression of the disease is halted), or incidence of engraftment failure. Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or a mammal) and includes: (1) inhibiting the disease, e.g., preventing engraftment failure; or (2) relieving the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means an amount which, when administered to a mammal in need thereof, is sufficient to result in a treatment or therapeutic benefit for that disease. Efficacy of an agent can be determined by assessing physical indicators of, for example, DA neuron engraftment, such as, e.g., tremor, bradykinesia, flexed posture, balance and coordination, etc. In some embodiments, engraftment or neural function may be measured in vivo (e.g., in humans) using a PET scan to measure metabolism or activity or dopaminergic systems (e.g., using PET tracers for imaging of the dopaminergic system). Efficacy can be assessed in animal models of Parkinson's disease, for example, by performing behavioral tests, such as step tests or cylinder tests.
[0144] C. Distribution for Commercial, Therapeutic, and Research Purposes
[0145] For purposes of manufacture, distribution, and use, the neural cells such as midbrain DA neurons as described herein may be supplied in the form of a cell culture or suspension in an isotonic excipient or culture medium, optionally frozen to facilitate transportation or storage.
[0146] Neural cells described herein may be provided different reagent systems, e.g., comprising a set or combination of cells that exist at any time during manufacture, distribution, or use. The cell sets may comprise any combination of two or more cell populations described in this disclosure, exemplified but not limited to programming-derived cells (neural lineage cells, their precursors and subtypes), in combination with undifferentiated stem cells or other differentiated cell types. The cell populations in the set may share the same genome or a genetically modified form thereof. Each cell type in the set may be packaged together, or in separate containers in the same facility, or at different locations, at the same or different times, under control of the same entity or different entities sharing a business relationship.
Therefore, the specification provides generic contemplative guidance to a method that focuses on treating parkinsonism that administer differentiated or stem cells locally or systemically to provide dopaminergic neural cells derived from human iPSC to a subject, particularly human in need thereof.
Working Examples: The specification described of the following (citations for pre-grant publication):
Example 1
[0148] Midbrain neuronal differentiation of human induced pluripotent stem cell (iPS) cell lines expanded on VTN-TN in Essential 8 medium was performed with small molecule and growth factor induction using a variety of differentiation media compositions and schedules as detailed in Tables 1-5. Generally, the iPS cells were cultured in D1 DA Neuron Induction Medium on Day 1, D2 Neuron Induction Medium on Day 2, and D3-D4 DA Induction Medium on Day 3 and 4. On Day 5, the cells were dissociated with TrypLE for 15 minutes and collected in DA Quench Medium before transferring the cells to a spinner flask suspension culture to form aggregates in D5 DA Neuron Aggregate Formation Medium.
[0149] On Day 6, the aggregates were settled, about 66% of the medium was removed, and the aggregates were fed DA Neuron Induction Medium. On Days 7-16, the aggregates were fed daily with DA Neuron Aggregate Maintenance Medium, and the medium was changed on Day 11 through 16. On Day 17, aggregates were dissociated to a single-cell suspension with TrypLE and plated onto Matrigel in D17 DA Neuron Aggregate Plating Medium. On Days 18, 20, 22 the medium was replaced with DopaNeuron Maturation Medium. On Day 24, the cells were dissociated using Accutase and plated in DA Neuron Maturation Plating Medium. The next day, the medium was replaced with DopaNeuron Maturation Medium.
[0150] On Days 27 and 29, the media was replaced with DA Neuron Maturation Medium plus Mitomycin C. On Day 31, the cells were dissociated with Accutase and re-plated onto poly-L-ornithine (PLO)/Laminin-coated flasks in DA Neuron Maturation Plating Medium. Next, on Days 32, 34, and 36, the cells were fed DopaNeuron Maturation Medium. On Day 37 or 38, the cells were again dissociated with Accutase and subjected to analysis or cryopreserved for later use.
Example 2
Efficient mDA Progenitor Patterning Using Mono-SMADi
[0151] Efficient patterning of mDA progenitors, as measured by the percentage of cells co-expressing FoxA2 and Lmx1 on process day 17, is generally required for obtaining a highly enriched population of mDA neurons at the end of the manufacturing process. If the majority of the cells on day 17 are not mDA progenitors, the neurons obtained will have a large population of non-midbrain phenotype neurons, or will have an outgrowth of proliferative cells that typically leads to neuron detachment or difficulties or an inability to purify the post-mitotic neurons.
[0152] The iCell DopaNeurons process was optimized with most progenitor patterning factors added starting on day 1. This includes factors for neuralization (dual-SMADi), sonic hedgehog signaling (Shh, PMN), and Wnt signaling (CHIR). However, the inventors found that this approach did not work well for almost all iPSC lines tested in the context of neuralization using mono-SMAD inhibition. A large number of optimization experiments were carried out across several iPSC lines to define conditions for efficient mono-SMADi mDA progenitor patterning, varying the timing and dose of all patterning factors. As shown in FIG. 1, conditions were found (“MONO/ALT”) where mono-SMADi mDA progenitor patterning was as good as dual-SMADi patterning using previously-optimized standard conditions (“DUAL/STD”), and the patterning was markedly more efficient than mono-SMADi mDA progenitor patterning using the standard conditions (“MONO/STD”). In the alternate (ALT) method, the SHH/Purmophamine addition was moved from Day 1 to Day 2. The changes utilized in this improvement included: (i) staggering the addition of the Wnt agonist (CHIR) to day 2 or day 3, (ii) re-optimizing the CHIR concentration, as this is dependent on the time it is first added, and (iii) moving the window where PD03 is added, as the optimal window depends on when CHIR is added. The end result was a narrow set of conditions that led to highly-efficient mDA progenitor formation in multiple iPS cell lines. Approximately half of the iPSC lines tested were able to generate mDA progenitors using mono-SMADi conditions. FIG. 2 shows an example of a matrix optimization experiment used to define optimized patterning conditions.
[0153] As shown in FIG. 1, the patterning of mDA progenitors from three different iPSC lines was evaluated on process day 17 by measuring the proportion of total cells expressing FoxA2 (by flow cytometry) or co-expressing FoxA2 and Lmx1 (by ICC). FoxA2/Lmx1 coexpression is the most accurate marker for mDA progenitor cells. The use of alternate patterning conditions allows for efficient mDA progenitor patterning in the context of mono-SMAD inhibition (“Mono/Alternate”), whereas the standard patterning conditions have poor mDA progenitor patterning in the context of mono-SMAD inhibition (“Mono/Standard”). Control differentiations using dual-SMADi and standard patterning conditions (“Dual/Standard”) are shown for comparison.
[0154] FIG. 1 data represents data obtained from using the conditions described in Table 1 for all Dual Standard conditions. Cell line C data was obtained using conditions described in Table 4 for Mono Alternate and Table 3 for Mono Standard conditions. Cell lines D and K followed Table 2 and Table 5 for Mono Standard and Mono Alternate, meaning that data from cell line C was obtained from testing LDN while the other lines were tested with Dorsomorphin.
[0155] Once an alternate patterning condition was identified that allowed for successful differentiation using mono-SMAD inhibition (Condition 1, i.e., Table 4: Alternate, LDN at 200 nM), the method was refined using a matrix of conditions, including the concentration of CHIR, and the treatment windows for Shh signaling, Wnt signaling, and MEK inhibition. As shown in FIG. 2, the following conditions were tested for mono-SMAD inhibition, as shown in Table 6 below. Table 6 shows the start day for incubation with a particular compound (e.g., “D2” indicates incubation start on day 2) and the concentration of CHIR99021 used. Shown in FIG. 2 is an example of one matrix experiment for iPSC line K. Conditions 3, 5, 7 and 9 resulted in the highest percentage of FoxA2+/Lmx1+ mDA progenitor cells by day 17, with the FoxA2+ population being maintained to day 24. Tyrosine hydroxylase (TH) expression is low in immature mDA neurons. These experiments were repeated using a concentration of CHIR99021 of 1.65 μM instead of 1.75 μM, and it was observed that similar results for the generation of mDA progenitor cells could be obtained using the lower 1.65 μM concentration of CHIR99021.
[0156] These mono-SMAD experiments were repeated, with the modification that Benzonase® (endonuclease, EMD Millipore) was included in the incubation on Day 5 at a concentration of 100 U/mL. Inclusion of the Benzonase® in the incubation on Day 5 was observed to reduce or prevent excessive clumping in the aggregate formation.
Example 11
Engraftment of iPSC-DA Neurons in Non-Human Primates
[0170] iPSC line "K" was differentiated to process completion (day 37) using the optimized protocol ("Condition 9") and cryopreserved. Cells were thawed and transplanted bilaterally to the caudate and putamen (1.5.times.10.sup.6 cells/injection) of MPTP-treated and immunosuppressed African Green Monkeys (n=3). After 3 months, engraftment and innervation of the neurons was assessed by histology of coronal sections. Staining of human cytoplasm (STEM121) reveals good human cell engraftment and innervation in all animals. Dopamine neurons (TH+) were observed in the grafts, demonstrating the ability of these cells to survive long-term and innervate the mDA neuron target structure in the brain. No tumors, neural outgrowth, or other adverse effects were observed in these animals. Results are shown in FIG. 13.
Example 12
Engraftment of iPSC-DA Progenitors and Neurons in Rat Parkinson's Disease Model System
[0171] iPSC line "K" was differentiated using the optimized mono-SMADi protocol ("Condition 9") and cryopreserved at different stages of the differentiation process (Day 17, day 24, and Day 37). In addition, iPSC-mDA cells derived using the optimized dual-SMADi protocol (iCell Dopa) were cryopreserved on process day 37. Cells were thawed and transplanted bilaterally to the striatum (4.5.times.10.sup.5 cells/injection) of 6-OHDA-treated but asymptomatic nude (RNU) rats (n=3 per group). After 3 months, engraftment and innervation of the cells was assessed by histology of coronal sections. Although neuron engraftment and innervation was observed in all four groups (Human NCAM stain), the iPSC-DA progenitor cells (day 17) and immature mDA neurons (day 24) had much larger grafts and greater innervation compared to the more mature mono-SMADi and dual-SMADi-derived mDA neurons (day 37 and day 37 iCell Dopa, respectively). In addition, larger numbers of DA neurons (TH+) were observed in the progenitor and immature DA neuron grafts. Ki67 staining revealed almost no proliferative cells in the grafts from day 37 cells, and few Ki67.sup.+ cells in the grafts from day 17 and day 24 cells. No tumors, neural outgrowth, or other adverse effects were observed in any of these animals. These results suggest that cells drawn from earlier in the optimized mono-SMADi differentiation process (day 17-24) are better able to engraft and innervate compared to more mature cells. Results are shown in FIG. 14.
Thus the working examples more narrowly describe a method of differentiating one iPSC cell for a varying number of days to arrive at three different sub-populations of cells: iPSC-DA progenitor cells (day 17), immature mDA neurons (day 24), and mature mDA neurons (day 37). The specification further states, “Efficient patterning of mDA progenitors, as measured by the percentage of cells co-expressing FoxA2 and Lmx1 on process day 17, is generally required for obtaining a highly enriched population of mDA neurons at the end of the manufacturing process. If the majority of the cells on day 17 are not mDA progenitors, the neurons obtained will have a large population of non-midbrain phenotype neurons, or will have an outgrowth of proliferative cells that typically leads to neuron detachment or difficulties or an inability to purify the post-mitotic neurons.” However, the neither the method of the claims nor the specification provides specific guidance to what percentage of cells that co-express FoxA2 and Lmx1 on day 17 is required for provide the requisite highly enriched population to obviate the difficulties described by the specification. The claims encompass delivering the differentiated cell population at any stage of the differentiation with number of FOXA2+/LMX1+ cells. However, the specification teaches that differentiated population produced prior to day 17 and prior to some requisite number of highly enriched FOXA2+/LMX1+cell will fail to predictably provide the requisite cell population necessary for its further use. Thus the specification expressly states that all the FOXA2+/LMX1+ cells will not predictably be usable in methods such as engraftment or treatment of parkinsonism. The specification and working examples provide guidance to methods that differentiate human iPSC into DA neuronal cell populations and administering these populations to animal models by local administration. They also provide guidance to engraftment studies. However, the specification and working examples fail to provide specific guidance to a method that treats any symptoms of any parkinsonism in subject. The specification and working examples fail to provide specific guidance to any methods that indirect administer the differentiated cells to the brain of such a subject. The specification and working examples fails to provide specific guidance to a method of providing requisite population of the differentiated Fox2A and LMX1 positive cell that will predictably treat parkinsonism. The specification fails to provide specific guidance to a method of providing a therapeutic amount of cells as the method requires. As such, the specification and working examples fails to provide enabled for the claimed therapeutic method.
State of the Art:
Regarding treating a parkinsonism (i.e. any or all symptoms including curing parkinsonism), Hiller et al (2022; of record in IDS 4/1/2022) states, “Current therapies are symptomatic, mostly focused on ameliorating motor deficits. Since no current therapy arrest or reverses the disease process, there is a major unmet need for new and effective PD treatments.” See page 1, paragraph 1.
Thus Hiller et al teaches that at the time of the effective filing date and even after, no therapies have been provided that reduce, alleviate, arrest, or reverse all symptoms of parkinsonism and most of the therapies. Even as much as six years after the effective filing date of the instant application most of the therapies being developed are targeted to reducing and/or alleviating motor function deficits. As such, the art teaches that embodiment of the claims directed to treating all and any symptoms of parkinsonism are not enabled at the time of effective filing and continuing post-filing.
Regarding DA neuron cell therapy, Hiller states, “A large body of work has demonstrated that rodent and human fetal ventral mesencephalic (hfVM) dopamine neurons survive well, innervate the host and form synapses, release dopamine, and alleviate motor deficits when grafted to the dopamine-depleted striatum of experimental animals... Some patients in open-label hfVM trials”® exhibited clinical improvement. However, randomized double blinded, placebo-controlled, clinical trials indicated that these benefits were too variable to meet the trials’ primary endpoints, although predefined secondary endpoints (Unified Parkinson's Disease Rating Scale, UPDRS) showed statistically significant benefits in younger (<60 years of age;…) or less impaired (UPDRS in off <49;…) subjects. Additionally, some patients developed graft-induced dyskinesias (GID)…, possibly related to pre-existing L-DOPA-induced dyskinesias and the transplants containing serotonergic cells alongside the desired dopaminergic neurons…. These findings prompted a re-evaluation of the approach. More recently, the European collaborative consortium, TRANSEURO, revisited fetal transplantation in an open-label trial (NCT01898390) with 11 patients at relatively early disease stages who had not developed significantL-DOPA-induced dyskinesias prior to grafting…”, See p. 1, paragraph bridging col 1 and 2).
These teaching from Hiller demonstrate that while DA neuron engraftment is well tolerated and the neurons are functioning in physiologically relevant ways, the cell therapies are not predictably arriving at therapeutic impact upon symptoms of parkinsonism, at the time of effective filing and continuing post-filing at least 6 years later. Thus, the art teaches that DA neuron engraftment does not consistently and predictably lead to a therapeutic effect and that overall DA neuron cell therapies are unpredictable.
As such, while Example 11 of the instant application provide promising results for engraftment in line with the teachings of the art of Hiller, the specification fails to further provide any specific guidance to arrive at a population of iPSC- DA neurons that predictably engraft, comprising a physiologically relevant phenotype, and art therapeutic to treat parkinsonism. Thus the guidance in the instant specification does not overcome the art-described obstacles and unpredictabilities to DA neuronal cell therapy for treatment of parkinsonism.
Hiller et al 2022 uses a modified version of the mono-SMAD method described in example 1 of the specification (p. 2, col 1, paragraph 1 under results section; . DA neuronal differentiation method and administers the differentiated cells at various days (i.e. stages) of differentiation (D17, D24, D37, or G418 cells) to the parkinsonism rat model used in the specification. These cells have not been subjected to any type of selection procedure to remove proliferative cells. The specification of instant application describes such a selection using G418 is used in their differentiation methods. The methods section further states that a non-engineered iPSC line that had been reprogrammed and expanded into a mater cell bank and a working cell bank were used. The iPSC-mDA differentiation protocol was adjusted for this iPSC line, including simplification of SMAD signaling inhibition (200 nM LDN-193189) and shifting GLK-3 inhibition (1.65 microM CHIR99021) 1 day later to process day 2 at a higher concentration adjusted for timing. Day 17 progenitors were manufactured using the same differentiation process, except the progenitor aggregates were dissociated with CTS TrypLE Select Enzyme and cryopreserved without exposure to maturation medium and mitomycin C treatment. (p. 12, col 2, cell differentiation section).
Hiller teaches that Hemiparkinsonian rat that received vehicle or D37 graft fails to demonstrate functional recovery. A mixed-effects ANOVA with Tukey’s post hoc testing revealed that rats receiving D17, D24, or G418 cells exhibited significant (P < 0.005; P < 0.005; P < 0.05) recover of motor asymmetry by 6 months post-injection. Additionally animal receiving D17 grafts displayed full normalization of rotations by 4 months post-injection (p. 3, paragraph bridging col 1 and 2).
As such, the post-filing art teaches that further characterization, selection, and modification of the mono-SMAD method was required to determine if any of the different cell type produced in the differentiation method would effectively treat one symptom of parkinsonism, which is the motor disfunction and rigidity. Further, these further modification and characterizations demonstrated that not all cell type produced by the claimed method will predictably treat motor asymmetry and rigidity. Particularly D37 mature DA neuron grafts did not demonstrate functional recovery. Therefore, the specification teaches that in the mono-SMAD method for less than D17 are unpredictable and comprise non-DA neural cells and other obstacle and the post-filing art demonstrates that only few subsets the differentiated cell population (D17 and D24) produced in a small window of time effectively alleviate the symptom of motor asymmetry. Further, the art teaches a significant degree of discovery experimentation was needed to arrive at subpopulation produced by the a variant of differentiation method of the application post-filing that solely treats one symptoms of parkinsonism. The specification does not provide specific guidance to selection of said subpopulation or that this subpopulation of cells will effectively treat parkinsonism in any way. The specification and art at the time of effective filing and post-filing do not provide guidance to treatment of any other symptoms of parkinsonism other than motor asymmetry and this is taught in the post-filing art. As such, the method of claims lacks enablement because at the time of effective filing the specific guidance to what stage of development and which subset of cells in the differentiation method result in cell capable of treating any symptoms of parkinsonism. Further neither the specification nor the art provide specific guidance to any symptom other than motor asymmetry that is treated by iPSC-Da using the mono-SMAD method. Even further, the specification does not provide any guidance to any of the post-filing alterations to the mono-SMAD method described by Hiller et al. that arrive at a subset of cell produced can treat one symptom of parkinsonism. As such, the neither the specification nor the art at the time of effective filing and post-filing enable the claimed method.
Regarding tumor formation by iPSC derived cells:
Lee et al. (PNAS 110(35):E3281-E3290, 2013) states, “The unique properties of human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) [i.e., indefinite self-renewal in vitro while maintaining their ability to differentiate into all cell types of the body upon exposure to relevant differentiation signals (1–3)] make them the best potential cell source for cell-based regenerative therapy and/or personalized medicine (4). Thus, enormous efforts have been undertaken to establish hESC- and hiPSC-based therapies for a variety of degenerative diseases (4–7). However, there are major technical and scientific obstacles remaining to be overcome before hPSC-based cell therapy becomes a realistic therapeutic modality. Most of all, it is of utmost importance to avoid possible teratoma/ tumor formation that can arise from any remaining undifferentiated pluripotent stem cells present in the differentiated cell mixture (8). Indeed, a systematic transplantation study demonstrated that the teratoma-forming propensity of various mouse iPSC-derived neurospheres correlated with the persistence of residual undifferentiated cells (9). Because hESCs and hiPSCs also exhibit marked variations in differentiation efficiencies (and remaining undifferentiated cells) (10–13), it is critical to remove all residual hiPSCs with teratoma potential before their clinical application. Despite numerous attempts at blocking teratoma formation, including introduction of suicide genes (14) or selecting the desired cell type (15), immunodepletion (16), or introducing cytotoxic antibody (17), a clinically viable strategy to eliminate teratoma formation remains to be developed (8,18)”. See p. E3281, paragraph bridging col 1 and 2.
Thus the art of Lee teaches at the time of the invention (2005) and long after (at least 2013), differentiated cells derived from pluripotent cells failed to be used for any type of cell therapy to treat a disorder in a patient because a means of predictably providing a homogenous differentiated cell population that predictably eliminates undifferentiated cells in the population had not been achieve. Lee highlights the absolute need for a homogenous population the differentiated cell type. A problem that can arise from have a heterologous population of ESC/iPSC-derived somatic cells and undifferentiated cells is tumor formation from the undifferentiated cell, thus causing a non-therapeutic effect, but further, a mixed population causes unpredictability in determining a therapeutic dose of cells because the variability in an unknown number and non-known character of the undifferentiated cell population.
Similar to Lee, Moon (Moon et al. International Journal of Stem Cells 4(1):24-34, 2011) teaches that a major problem involving the use of hESC is the possibility of cell misbehavior following transplantation. This potentially serious complication can occur if any transplanted undifferentiated hESC form teratomas. Careful and previse protocols for collecting only differentiated cells are necessary to circumvent this problem. Moon further teaches also. As of 2011 researchers still needed to develop way to screen pluripotent cell derivatives for contamination with undifferentiated cells and derivative cells that have optimized chromosomally stable induction systems in vivo (p.30, col 1, past full paragraph starting with, “Another major risk…”; paragraph bridging col 1 and 2 of pp. 31). Moon further teaches that during prolonged maintenance of undifferentiated pluripotent cell in vitro, chromosomal instability gives rise to numerous changes in ploidy, especially chromosomes 13 and 17. Moon states, “Chromosomal instability seriously affects cell function and is well known to be a hallmark of many tumor. In addition, the generation of chromosomal instability by the presence of extra centrosomes can give rise to multiple aneuploidy daughter cells with proliferative advantages, chemoresistance, or metastatic potential (66, 67). Their potential for tumor progression is a significant challenge facing the safe therapeutic application of hESC, Recently, Moon et al. demonstrated aberrant cell division in karyotically abnormal hESC during in vitro culture. These authors subsequently show a post-transplant tumor-like growth arising from the hESC transplant derivative (68). These results emphasize that sustaining normal human chromosome number during in vitro culture and differentiation is indispensable for safe transplantation of hESC derivatives. For therapeutic use”.
Regarding chromosomal instability, Lee teaches accumulation of mutations in pluripotent cells would result in enormous harmful effects to all subsequent derived somatic cells arising at later developmental stages and/or those of further generations. Thus, it is critical that these cells maintained their genomic integrity by establishing unique mechanisms, such as significantly lower mutations rates and lower frequencies of mitotic mutations than their differentiated counterparts (E3288, col 1, paragraph 1 under ‘Discussion’ section.)
Thus Moon and Lee both highlight the obstacle of chromosomal instability in pluripotent stem cells and somatic cell differentiated from them. Both describe not only how enormously harmful these chromosomally instable cells can be to the safety someone receiving these cells, but the grave degree of cellular dysfunction and change of cellular function that occurs in these cell derivatives. Thus, not only are they describing a hindrance to safe therapeutic use of differentiated cell derived from pluripotent cell, they are describing a lack of predictable function (i.e. function as the intended differentiated cell type) because the genomic instability leads to cellular dysfunction.
Similar to the prior art of Lee and Moon, Bedel (Bedel et al. Stem Cells Translational Medicine 2017; 6:382-393) reports, “we and others have developed potent protocols for in vitro hematopoietic differentiation of stem cells after they have been genetically corrected [3–5]. However, several aspects hinder the progression of iPSCs into clinic. In particular, for hematopoietic applications, there is no current method of differentiation yielding a 100% pure population from a pluripotent donor source with regard with hematopoietic differentiation [4]. Therefore, a population of differentiated cells is always contaminated by residual undifferentiated iPSCs. This becomes a critical hurdle to the application of iPSCs in clinical protocols because of their iatrogenic teratogenesis potential [6].” See paragraph bridging pp. 382-383.
Thus Bidel demonstrates that the obstacle of provide a pure population of pluripotent stem cell derived cells that did not form teratoma still was not established post-filing and that the obstacles described in Lee and Moon persist.
Thus, the art teaches that at the time the claimed invention was made and after, the use of cell differentiated from pluripotent stem cells for therapy had not been established due to complications of the presence of undifferentiated cells and cells with genomic instability. The art teaches not only a hindrance to the safe use of these cells for therapeutic purposes but also concern and obstacle to correct, predictable function of the pluripotent cell derived somatic cells. Thus, the art teaches that the use of these cells in therapy methods as claimed is highly unpredictable.
The post-filing art of Hiller and Example 11 teach that the mono-SMAD method, result in proliferative cells in vivo following administration. However, both also teach that the proliferative cells are not associated with tumor formation, which suggests that subpopulation provided by applicant’s method can be used for in vivo purposes. However, post-filing art of Hiller demonstrates the claimed method results in mixed cell populations including proliferative cells that are not the target, therapeutic cell. Further, these resultant cell population cell type composition varies depending on what day in the differentiation method and when the factors are being added to the culture. Neither the specification nor the art provide guidance to which cells in the various time and factor dependent method result in a cell that is therapeutic and which cells are actually the therapeutic cells. As such, the presence of cells that are undifferentiated and the yet unidentified cell in the mixed population are imparting a therapeutic effect remain problematic for determining “a therapeutically effective amount” of cells as the claims recite.
Amount of Experimentation: Hiller provides some evidence that post-filing experimentation would be needed to enable the instantly claimed method at least by one species embodiment of the claimed method (i.e. D17 cells alleviating motor asymmetry). However, Hiller teaches that modifications to the mono-SMAD method and selection methods and discovery of only a small subset of cells that are potentially therapeutic to motor asymmetry. This determination of a select cell population that arrives on D17 that is capable of alleviating motor asymmetry is discovered post-filing using a modified version of the methods described by the specification. This type of modification and discovery is not just routine optimization of the parameters disclosed in the specification but new essential information and methodology required to enable the claimed method. As such, the method described by the specification is not enabled at the time effective filing as required but rather only enabled post-filing by adding new findings and methods which is not permitted. Thus the amount of experimentation at the time of effective filing would be deemed undue.
Therefore, the instantly claimed invention lack enablement because the specification fails to provide specific guidance to a therapeutic method that provides a population of FOXA2/LMX1 positive cells derived from pluripotent stem cells that is a therapeutically effective amount to treat a parkinsonism. Further, the art at the time of the effective filing fails to supplement the shortcomings of the specification. Further, the amount of experimentation required to overcome the unpredictabilites described in the art goes beyond routine experimentation into discovery of new techniques and technologies. Thus, the amount of experimentation to determine if the claimed method can be enabled is undue.
Therefore, at the time of filing the skilled artisan would need to perform an undue amount of experimentation without a predictable degree of success to implement the invention as claimed.
Response to Arguments
Applicant's arguments filed 1/27/2026 have been fully considered but they are not persuasive.
Applicant submits the amendments to the claims make the rejection of record moot. Applicant submits that Examiner indicates that the application provides support for treating Parkinson’s disease.
In response, Examiner did not indicate that the specification provide “support” or enablement for treatment of Parkinson’s Disease (PD). To the contrary the rejection of record expressly states that treatment of any form of parkinsonism is not enabled by the specification and prior art.
Applicant submits that the Examples and the post-filing data in Hiller et al provide clear support for treatment of PD with dopaminergic progenitor cells as claimed. Applicant submits that the claims require culturing in the presence of modulators for a sufficient amount of time to provide a cell composition with 50% progenitor cells and the cells are administered to a region of the basal ganglion with the post-filing art of Hiller demonstrating treatment of PD.
In response, as previously stated in prosecution, the examples provides some enabling support for engraftment and innervation produced form administering the progenitor cells. However, engraftment and innervation are NOT “treatment” or therapeutic. The examples do not demonstrate elevation of symptoms of parkinsonism and as such fails to enable the recited result of treatment. Regarding Hiller, as previously made of record, their methodology is a post-filing modification of a method similar to the method disclosed in the application and is not commensurate in scope. Further Hiller describes post-filing improvements that are not described by the instant application and thus would not be considered enabling at the time of the invention as required.
Thus Applicant’s arguments and amendments are not found persuasive in enabling the instantly claimed method for treatment of PD as claimed. Therefore, the rejection is maintained and applied to the new claims for reasons discussed above and previously made of record.
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MARCIA STEPHENS NOBLE whose telephone number is (571)272-5545. The examiner can normally be reached M-F 9-5:30.
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MARCIA S. NOBLE
Primary Examiner
Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632