	US 12,241,069 B2
	Gas vesicle expression systems, gas vesicle constructs and related genetic circuits, vectors, mammalian cells, hosts, compositions, methods and systems
Arash Farhadi, Pasadena, CA (US); Gabrielle H Ho, Pasadena, CA (US); Daniel P Sawyer, Pasadena, CA (US); Mikhail Shapiro, Pasadena, CA (US); Robert C. Hurt, Pasadena, CA (US); and Mengtong Duan, Pasadena, CA (US)
Assigned to CALIFORNIA INSTITUTE OF TECHNOLOGY, Pasadena, CA (US)
Filed by CALIFORNIA INSTITUTE OF TECHNOLOGY, Pasadena, CA (US)
Filed on May 15, 2023, as Appl. No. 18/317,915.
Application 18/317,915 is a continuation of application No. 16/736,683, filed on Jan. 7, 2020, granted, now 11,761,008.
Claims priority of provisional application 62/789,295, filed on Jan. 7, 2019.
Claims priority of provisional application 62/895,553, filed on Sep. 4, 2019.
Prior Publication US 2023/0357780 A1, Nov. 9, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/67 (2006.01); C07K 14/195 (2006.01)

CPC C12N 15/67 (2013.01) [C07K 14/195 (2013.01); C12N 2510/02 (2013.01)]

32 Claims

1. A method of ultrasound imaging to be used on a target site comprising a mammalian cell, the method comprising:

introducing into the mammalian cell a Gas Vesicle Expression System (GVES) configured of one or more gene clusters of gvp genes (GVGC) encoding GV proteins capable of forming a GV type having an acoustic collapse pressure threshold, the Gas Vesicle Expression System comprising:

a GVPA/B gene expression cassette comprising a gvpA or a gvpB gene under control of a mammalian promoter and additional mammalian regulatory regions in a configuration allowing expression of the gvpA or gvpB protein in the mammalian cell; and

one or more additional gvp gene expression cassettes comprising the gvp genes of the GV gene cluster other than the gvpA and gvpB, under control of a mammalian promoter and additional regulatory regions in a configuration allowing expression of the GV proteins other than the gvpA and gvpB in the mammalian cell,

wherein each of the one or more additional gvp gene expression cassettes, when comprising two or more gvp genes, further comprises a separation element between the two or more gvp genes configured to provide a separate expression of the corresponding GV protein;

wherein the GVPA/B gene expression cassette and the one or more additional gvp gene expression cassettes are operably linked by regulatory sequences allowing co-expression of the GV proteins and formation of the GV type in the mammalian cell, and

wherein the one or more gas vesicle gene clusters comprise at least one of

a GVGC having gvpB, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, and gvpU from B. megaterium;

a GVGC having gvpA, gvpC, gvpN, gvpJ, gvpK, gvpF, gvpG, gvpV, and gvpW from Anabaena flos-aquae;

a GVGC having gvpR, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT and gvpU from B. megaterium and gvpA gene from Anabaena flos-aquae;

a GVGC having gvpA, and gvpC from Anabaena flos-aquae, and gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, and gvpU from B. megaterium; and

a GVGC having gvpA, gvpC and gvpN from Anabaena flos-aquae, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, and gvpU from B. megaterium,

the introducing being performed for a time and under conditions to allow expression of the GV proteins and the production of one or more GV types, having the acoustic collapse pressure threshold, in the mammalian cell;

the method further comprising:

applying ultrasound to the target site at a peak positive pressure less than the acoustic collapse pressure threshold;

increasing peak positive pressure (PPP) to above the selective acoustic collapse pressure value threshold as a step function;

imaging the target site in successive frames during the increasing;

extracting a time-series vector for each of at least one pixel of the successive frames; and

detecting from the time-series vectors a transient signal from, due to the increasing PPP, fluid displacement from collapsing of the GVs or cavitating bubbles released from the GVs, the detecting being in a time domain of the successive frames, the transient signal providing an increase in contrast signal in the ultrasound imaging.