Neural cells expressing adenovirus E4ORF1, and methods of making and using the same
Inventors
Qiang, Liang • Nolan, Daniel Joseph • Davis, Claude Geoffrey
Assignees
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Publication Number
US-10947500-B2
Publication Date
2021-03-16
Expiration Date
Abstract
In certain aspects the present invention provides engineered neural cells, neural stem cells, or neural progenitor cells that contain a nucleotide sequence that encodes an adenovirus E40RF1 polypeptide and/or that contain an adenovirus E40RF1 polypeptide. The present invention also provides methods of making and using such engineered cells and compositions comprising such engineered cells.
Core Innovation
Engineered neural cells, engineered neural stem cells, and engineered neural progenitor cells are provided as substantially pure isolated populations. The engineered cells comprise a nucleic acid sequence that encodes an adenovirus E4ORF1 polypeptide operably linked to a promoter, and the engineered cells do not comprise an entire adenovirus E4 region or E4ORF2, E4ORF3, E4ORF4, E4ORF5, or E4ORF6 coding sequences or amino acid sequences.
The disclosure further provides in vitro engineered blood brain barrier model system compositions that comprise endothelial cells and a population of engineered glial cells. The engineered glial cells comprise a nucleic acid sequence that encodes an adenovirus E4ORF1 polypeptide operably linked to a promoter and do not comprise an entire adenovirus E4 region or E4ORF2, E4ORF3, E4ORF4, E4ORF5, or E4ORF6 coding sequences or amino acid sequences. The nucleic acid sequence is carried by a viral vector selected from lentiviral, retroviral, adeno-associated virus, or herpesvirus vectors.
Neural cell compositions are also provided for delivery to a subject. The in vitro composition comprises neural cells and a carrier solution, wherein at least 75% of the neural cells are engineered neural cells that comprise a nucleic acid sequence encoding an adenovirus E4ORF1 polypeptide and do not comprise an entire adenovirus E4 region or any E4ORF2–E4ORF6 coding sequences or amino acid sequences. The disclosure further includes culturing methods for neurons comprising co-culturing neurons with engineered glial cells or culturing neurons with glial-cell conditioned medium obtained from engineered glial cell cultures.
A problem is addressed in the context of neural cells by using adenovirus E4ORF1 to support improved neuronal morphology and glial proliferation without dedifferentiation or transformation. Engineered astrocytes expressing E4ORF1 maintain GFAP+ astrocyte phenotype through multiple passages while showing increased cell numbers. Engineered hippocampal neurons expressing E4ORF1 show increased axon length, more minor processes, and more primary axonal branches while retaining neuronal markers including βIII tubulin, MAP2, tau, NeuN, and neurofilament.
Claims Coverage
The independent claims cover four core areas: an engineered neural cell population, an in vitro blood-brain barrier model system, an in vitro delivery composition with a carrier solution, and culturing neurons using E4ORF1-containing engineered glia or glial-cell conditioned medium. The inventive theme is the use of a nucleic acid encoding adenovirus E4ORF1 under an operably linked promoter while excluding an entire adenovirus E4 region and excluding E4ORF2–E4ORF6 coding sequences or amino acid sequences, with additional claim context for a blood-brain barrier model, delivery composition constraints, and neuron culturing format.
Engineered neural cell population with operably linked E4ORF1 and excluded E4ORF2-E4ORF6
A substantially pure isolated population of engineered neural cells, engineered neural stem cells, or engineered neural progenitor cells comprising a nucleic acid sequence that encodes an adenovirus E4ORF1 polypeptide operably linked to a promoter, wherein the engineered cells do not comprise an entire adenovirus E4 region and do not comprise any E4ORF2, E4ORF3, E4ORF4, E4ORF5 or E4ORF6 coding sequences or amino acid sequences.
In vitro blood brain barrier model system with engineered glia expressing E4ORF1 and excluded E4ORF2-E4ORF6
An in vitro blood brain barrier model system comprising endothelial cells and a population of engineered glial cells, wherein the engineered glial cells comprise a nucleic acid sequence that encodes an adenovirus E4ORF1 polypeptide operably linked to a promoter, and do not comprise an entire adenovirus E4 region and do not comprise any E4ORF2, E4ORF3, E4ORF4, E4ORF5 or E4ORF6 coding sequences or amino acid sequences.
In vitro delivery composition with carrier solution enriched for E4ORF1 engineered neural cells and excluded E4ORF2-E4ORF6
An in vitro composition comprising neural cells and a carrier solution for delivery to a subject, wherein at least 75% of the neural cells in the composition are engineered neural cells that comprise a nucleic acid sequence that encodes an adenovirus E4ORF1 polypeptide, do not comprise an entire adenovirus E4 region, and do not comprise any E4ORF2, E4ORF3, E4ORF4, E4ORF5 or E4ORF6 coding sequences or amino acid sequences.
Neurons cultured with E4ORF1-containing engineered glia or glial-cell conditioned medium
A method for culturing a population of neurons comprising either culturing the population of neurons and a population of engineered glial cells together in the same culture vessel, wherein the engineered glial cells contain a nucleotide sequence that encodes an adenovirus E4ORF1 polypeptide or contain an adenovirus E4ORF1 polypeptide, or contacting the population of neurons with glial-cell conditioned medium obtained from a culture of the engineered glial cells.
Across the independent claims, the patent coverage centers on engineered neural and glial cells carrying a nucleic acid encoding adenovirus E4ORF1 under an operably linked promoter while excluding an entire adenovirus E4 region and excluding E4ORF2–E4ORF6 coding sequences or amino acid sequences. This engineered system is applied to an in vitro blood-brain barrier model, an in vitro delivery composition with carrier solution and an engineered-cell proportion requirement, and neuron culturing using either co-culture with engineered glial cells or glial-cell conditioned medium.
Stated Advantages
Improves neuronal morphology, including increased axon length, more minor processes, and more primary axonal branches.
Promotes glial proliferation while maintaining GFAP+ astrocyte phenotype through multiple passages.
Supports neuronal and glial effects without dedifferentiation or transformation.
Documented Applications
Therapeutic regeneration/repair [application described in partial content summary].
In vitro blood-brain barrier model system comprising endothelial cells and engineered astrocytes and/or pericytes [application described in partial content summary].
Co-culture/feeder cell layer formats for culturing neurons with engineered glial cells [application described in partial content summary].
Conditioned-medium formats for culturing neurons using glial-cell conditioned medium obtained from engineered glial cells [application described in partial content summary].
In vitro compositions for delivery to a subject using a carrier solution [application described in independent claim 21].
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