Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10633689-B2
Publication Date
2020-04-28
Expiration Date
Abstract
Methods for measuring REP-1 and REP-2 activity are provided. In certain embodiments, a method includes: (a) contacting cells that do not express endogenous functional REP-1 or REP-2 protein with an adeno-associated viral (AAV) vector comprising a CHM gene encoding a REP-1 protein or CHM like gene encoding a REP-2 protein under conditions allowing cell transduction; (b) incubating transduced cells under conditions allowing expression of the encoded REP-1 or REP-2 protein; (c) lysing the transduced cells to produce an extract comprising the encoded REP-1 or REP-2 protein and Rab small GTPase (Rabs); (d) incubating said extract with a Rab substrate for a period of time and under conditions allowing prenylation of the Rab thereby forming prenylated Rab; and (e) detecting and/or quantifying the prenylated Rab, wherein the amount of prenylated Rab reflects REP-1 or REP-2 activity thereby measuring REP-1 or REP-2 activity.
Core Innovation
The invention is directed to measuring REP-1 activity using REP-1 negative RPE cells (CHMneg) that do not express endogenous functional REP-1 protein. REP-1 activity is measured by introducing a CHM gene encoding a REP-1 protein into CHMneg cells using an adeno-associated viral (AAV) vector under conditions allowing cell transduction and expression of the encoded REP-1 protein.
The transduced cells are lysed to produce an extract comprising the encoded REP-1 protein and Rab small GTPase (Rabs). The extract is incubated with a Rab substrate under conditions allowing prenylation of the Rab thereby forming prenylated Rab, where the amount of prenylated Rab is used as a functional readout of REP-1 activity.
Prenylated Rab is detected and/or quantified, and the measured amount of prenylated Rab reflects REP-1 activity. In the provided assay context, the prenylation substrate is described as a Rab prenylation substrate such as GGPP, including non-radioactive labeling and radioactive labeling options, with examples including western blot and WES automated immunoassay.
Claims Coverage
The provided independent claim set contains one independent claim, which specifies a complete workflow with five inventive elements: AAV-mediated introduction of a CHM gene encoding REP-1 into CHMneg RPE cells, expression of the encoded REP-1 protein, lysis to generate an extract containing encoded REP-1 protein and Rab small GTPase (Rabs), incubation with a Rab substrate to form prenylated Rab, and detection/quantification of prenylated Rab where the amount reflects REP-1 activity.
Measuring REP-1 activity using AAV-driven REP-1 expression in REP-1 negative RPE cells
A method for measuring REP-1 activity comprising contacting REP-1 negative RPE cells (CHMneg) that do not express endogenous functional REP-1 protein with an adeno-associated viral (AAV) vector comprising a CHM gene encoding a REP-1 protein under conditions allowing cell transduction, and incubating the transduced cells under conditions allowing expression of the encoded REP-1 protein.
Prenylation-based functional readout from a cell extract containing Rabs
A method further comprising lysing the transduced cells to produce an extract comprising the encoded REP-1 protein and Rab small GTPase (Rabs), and incubating said extract with a Rab substrate for a period of time and under conditions allowing prenylation of the Rab thereby forming prenylated Rab.
Detecting and/or quantifying prenylated Rab to reflect REP-1 activity
Detecting and/or quantifying the prenylated Rab, wherein the amount of prenylated Rab reflects REP-1 activity thereby measuring REP-1 activity.
Across the single independent claim, REP-1 activity is measured by AAV introduction of a CHM gene encoding REP-1 into CHMneg RPE cells, followed by expression, lysis to an extract containing encoded REP-1 and Rabs, prenylation of Rab substrate to form prenylated Rab, and detection/quantification of prenylated Rab as the functional readout of REP-1 activity.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Choroideremia assay context using iPSC-derived RPE isogenic controls (REP-1 positive and REP-1 negative) is discussed.
Interested in licensing this patent?