Positional delivery and encoding by oligonucleotides of biological cells for single cell sequencing (POS SEQ)

Techniques for positional delivery and position encoding by oligonucleotides of biological cells for single cell RNA sequencing are provided. In one aspect, a method of positional delivery and encoding of cells in a biological sample includes: encoding the cells in the biological sample for single cell sequencing by delivering molecular probes inside the cells that encode a position of the cells in the biological sample. A system for positional delivery and encoding of cells in a biological sample is also provided.

FIELD OF THE INVENTION

The present invention relates to single cell sequencing, and more particularly, to techniques for positional delivery and position encoding by oligonucleotides of biological cells for single cell ribonucleic acid (RNA) sequencing, i.e., positional sequencing (POS SEQ).

BACKGROUND OF THE INVENTION

The successful functioning of multi-cellular organisms relies on the coordinated functions of a multitude of molecular constituents from individual cells and the interactions among functionally distinct cells. Further, these molecular constituents are constantly changing such as in response to cell-to-cell interactions which oftentimes result from local physical cell-to-cell contact and/or from short length-scale paracrine cell-to-cell communications. Thus, the state of a biological system is often defined by the relative position of the cells in the system and the highly dimensional molecular composition of each of those cells.

For example, with diseases such as cancer, specific tumor cell subpopulations can co-opt adjacent normal cells to support tumor progression. Thus, the relevance of cell positioning has motivated the development of therapeutic agents that target the co-opted cells, such as platelet-derived growth factor receptor (“PDGFR”) inhibitors to target PDGFR+ pericytes, and small molecule inhibitors or neutralizing antibodies of colony-stimulating factor 1 (“CSF1”) receptors to target macrophages.

Typically, spatial and molecular measurements are made using image-based analysis where molecular and positional information is obtained by taking microscopy images of samples treated with either enzymatically- or fluorescently-labeled antibodies that bind specifically to the molecular target of interest. When the images are digital, the sensor pixel position reflects the spatial relationship of the cells, while the sensor pixel signal intensity reflects the local density of the labeled antibodies molecular target of interest.

Other techniques employed for concomitant spatial and molecular measurements involve first recording the positioning of the individual cells that are then measured. It is however impractical to implement such a technique with potentially millions of distinct cells that need to be stored and processed separately for molecular profiling.

Thus, improved techniques for concomitant spatial and molecular measurements of biological cells would be desirable.

SUMMARY OF THE INVENTION

The present invention provides techniques for positional delivery and position encoding by oligonucleotides of biological cells for single cell ribonucleic acid (RNA) sequencing (POS SEQ). In one aspect of the invention, a method of positional delivery and encoding of cells in a biological sample is provided. The method includes: encoding the cells in the biological sample for single cell sequencing by delivering molecular probes inside the cells that encode a position of the cells in the biological sample.

In another aspect of the invention, another method of positional delivery and encoding of cells in a biological sample is provided. The method includes: constructing a cDNA library of molecular probes that encode a position of cells in a biological sample; linking the molecular probes to a vessel; delivering the vessel with the molecular probes to specific locations of the biological sample where the vessel delivers the molecular probes inside the cells at the specific locations; extracting the cells containing the molecular probes from the sample; and performing single cell sequencing of the extracted cells.

In yet another aspect of the invention, a system for positional delivery and encoding of cells in a biological sample is provided. The system includes: a processor device, connected to a memory, that is implemented to: analyze data from single cell sequencing of cells along with molecular probes, that have been delivered inside the cells, which uniquely encode a position of the cells in a biological sample.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Provided herein are techniques for concomitant positional and molecular measuring of cells using a molecular probe having a unique oligonucleotide sequence that encodes a positioning of the cells within a biological sample such as a cell culture (e.g., a cell culture including living eukaryotic and/or prokaryotic cells) and/or a tissue sample (e.g., a biopsy, formalin-fixed paraffin-embedded (“FFPE”) and/or frozen tissue containing living cells). Thus, when the cells are later dissociated from the biological sample and sequenced, the cells will take with them the positional information encoded in the molecular probe. Advantageously, the molecular probe encodes the position at which each of the cells being sequenced is located within the biological sample.

As will be described in detail below, the molecular probe is first linked to a vessel such as a retrovirus, disulfide-linked cell-penetrating peptide (CPP) and/or bead micro-particle. A liquid cargo delivery device such as a microfluidic probe (MFP) is then used to deliver the molecular probe/vessel to specific locations of the biological sample. See, for example, Juncker et al., “Multipurpose microfluidic probe,” Nature Materials, Advanced Online Publication (July 2005) (8 total pages), the contents of which are incorporated by reference as if fully set forth herein. By way of the vessel, the molecular probes with unique nucleotide sequences are delivered inside the cells at those specific locations of the biological sample. As highlighted above, these oligonucleotide sequences are in effect a label of the position of a given cell in the biological sample. Thus, for each location (x,y) of a biological sample that the liquid cargo delivery device visits, a unique oligonucleotide sequence is delivered inside the cells at that location in the biological sample.

An overview of the present techniques for positional delivery and encoding of cells in a biological sample for single cell sequencing is now provided by way of reference to methodology100ofFIG.1. In step102, a complementary deoxyribonucleic acid (cDNA) library of molecular probes containing unique oligonucleotide sequences is constructed. As will be described in detail below, according to an exemplary embodiment, the library construction leverages the template-switching activity of Moloney murine leukemia virus reverse transcriptase (“MMLV RT”). For a general description of MMLV RT for library construction see, for example, Zhu et al., “Reverse Transcriptase Template Switching: A SMART™ Approach for Full-Length cDNA Library Construction,” BioTechniques 30:892-897 (April 2001) (hereinafter “Zhu”), the contents of which are incorporated by reference as if fully set forth herein.

In step104, the molecular probes with unique oligonucleotide sequences are then linked to a particular vessel such as a retrovirus, coupled to a disulfide-linked cell-penetrating peptide (CPP) or a bead micro-particle. This vessel will enable the molecular probes to be delivered inside the cells of a biological sample. By delivering the molecular probes into the cells, the cells can be uniquely identified—even when disassociated from the biological sample—due to the unique oligonucleotide sequences carried by the molecular probes.

In step106, the vessels with the molecular probes are delivered to specific locations of the biological sample (e.g., a living cell culture and/or tissue sample with living cells), where the vessels deliver the molecular probes inside the cells at those specific locations. According to an exemplary embodiment, this location-specific delivery is accomplished using a liquid cargo delivery device such a microfluidic probe or MFP. A microfluidic probe is a non-contact, scanning platform that can hydrodynamically localize as little as 100 picoliters of a liquid cargo with micrometer precision. For instance, the molecular probes can be dispersed in a processing solution (e.g., an aqueous solution) that is then delivered via the liquid cargo delivery device to specific locations of the biological sample. The molecular probes delivered to a given specific location of the biological sample contain a unique oligonucleotide sequence that is associated with that given specific location. Thus, as provided above, when the cells are later disassociated from the biological sample for sequencing, the oligonucleotide sequence encodes the original position of the cells in the sample (i.e., positional encoding).

Once the molecular probes are delivered to a given specific location of the biological sample, the vessels deliver the molecular probes inside the cells at those specific locations. According to an exemplary embodiment, the cells take in the vessels with the molecular probes through an active transfection/transduction process using living cell machinery. Thus, the present techniques are preferably performed with a living biological system. For instance, the biological sample preferably contains living cells, whether as a living cell culture or as a tissue sample containing living cells. The living cells permit transfection/transduction to occur. Following the present positional encoding process, the cells/tissue can be fixed if so desired.

In step108, single cell sequencing is performed on the cells extracted from the biological sample. Even though the cells are disassociated from the biological sample for sequencing, the cells now contain the molecular probe with oligonucleotide sequence encoding the position of the cells in the biological sample. Thus, this positional data can be retained through the sequencing process.

For instance, in step110the data from the single cell sequencing is stored and analyzed (e.g., in silico) along with the data from the molecular probes which uniquely encodes the positions of the cells in the biological sample. An exemplary apparatus for storing and analyzing this data is provided inFIG.9, described below. Being able to analyze transcriptomic data (i.e., RNA transcripts produced by a genome) from the cells along with the position of those cells in the biological sample is extremely beneficial. For instance, as highlighted above, the state of a biological system is often defined by the relative position of the cells in the system and the highly dimensional molecular composition of each of those cells. Take, for example, the development of therapeutic agents for cancer treatment that leverage cell positioning to target specific tumor cell subpopulations. See above.

As described in conjunction with the description of step102of methodology100above, the process begins with the construction of a cDNA library of molecular probes containing unique oligonucleotide sequences for positional encoding. As shown inFIG.2, the cDNA library construction begins with many barcoded DNA oligonucleotide primer molecules204conjugated to a microparticle202(e.g., a bead) such as a glass bead. The techniques for preparing distinctly barcoded oligonucleotide primers are described generally in Macosko et al., “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets,” Cell 161, 1202-1214 (May 2015), the contents of which are incorporated by reference as if fully set forth herein.

As shown inFIG.2, beginning from its5′ end, each DNA oligonucleotide primer molecule204contains a universal polymerase chain reaction (PCR) handle204a, a cell barcode204b, a unique molecular identifier (UMI)204c, a position code204d, and a poly T sequence204e(i.e., Tn—a sequence of n thymine repeats). PCR handle204aenables PCR amplification. For example, according to an exemplary embodiment, PCR handle204ais a DNA oligonucleotide sequence for PCR primers in the amplification step (see, e.g.,FIG.4—described below).

Cell barcode204bis a DNA oligonucleotide sequence that is unique to bead202/cell into which the molecular probe is delivered. UMI204cis a DNA oligonucleotide sequence that is unique to this particular DNA oligonucleotide primer molecule204. For instance, the DNA oligonucleotide primer molecules attached to the same bead202can share the same cell barcode, but different UMIs. In other words, the UMIs of each DNA oligonucleotide primer molecules has a different, unique oligonucleotide sequence. By way of example only, the UMIs can be used for normalizing gene counts during computational data processing. For example, the UMIs can be used to identify PCR duplicates during the single cell sequencing (see below).

The position code204dprovides the (location-specific) oligonucleotide sequence for positional encoding. Namely, as described above, the position code204duses a unique oligonucleotide sequence to encode the location (x,y) of cells in a biological sample into which the present molecular probes will be delivered. A length of the position code204dcan depend on the total number of locations (x,y) to be encoded. For example, according to an exemplary embodiment, the length of the position code204dis determined as follows,
L≥log 4(N),  (1)
wherein L represents the length of the position code204d(i.e., the number of nucleotides that make up the position code204d), and wherein N represents the total number of locations (x,y) to be encoded. As will be described below, the library and/or library construction (such as the generation of the location-specific oligonucleotide sequence for positional encoding) can optionally be provided as a service in a cloud environment.

According to an exemplary embodiment, the cDNA library is constructed using MMLV RT. See, for example,FIG.3where an endogenous messenger RNA (mRNA) template302hybridizes with DNA oligonucleotide204, and MMLV RT304synthesizes a DNA complement (cDNA) to the mRNA template302, and then appends a poly cytosine (C) sequence to the newly synthesized cDNA sequence. For instance, as shown in step310, in its simplest form mRNA template302of the ithgene includes a generic5′ CAP302a, a gene-specific coding region gi302b, and a poly A tail302c(i.e., a sequence of m adenine (A) repeats).

As shown in step312, the mRNA template302hybridizes with the3′ poly T sequence204eof DNA oligonucleotide204, and the MMLV RT304synthesizes a DNA complement (see for example gene-specific coding region fi204f) to the mRNA template302. This new cDNA sequence is now given reference numeral204′. MMLV RT304then appends cDNA sequence204′ with poly C sequence204g.

According to an exemplary embodiment, a template switch is performed where a template switch oligonucleotide (TSO) sequence402is hybridized with the cDNA sequence204′, after which the MMLV RT304performs the ‘template switch’ in which MMLV RT304uses the TSO sequence402as a template for replication. SeeFIG.4. As shown inFIG.4, TSO sequence402includes two groups, an oligonucleotide code402athat one wants to append to the mRNA template302, and a poly ribosomal Guanine (rG) repeat sequence402b. According to an exemplary embodiment, the oligonucleotide code402ais a PCR handle. As provided above, a PCR handle enables PCR amplification.

As shown in step410, the poly rG sequence402bof TSO402hybridizes with the poly C sequence204g(that was appended to the cDNA sequence204′ by MMLV RT304—see above). Doing so enables the MMLV RT304to then use TSO402as a template for replication. For instance, as shown in step410MMLV RT304appends a PCR handle204hto the poly C sequence204gat the3′ end of cDNA sequence204′.

As shown in step412, the cDNA sequence204′ is then separated from the mRNA template302/TSO402. By way of example only, the cDNA sequence204′ can be separated from the mRNA template302/TSO402by ribonuclease H activity of the MMLV RT technology and/or through the use of RNA degradation by sodium hydroxide (NaOH) and heat. The result is a molecular probe with a unique oligonucleotide sequence (i.e., position code204d) that encodes positional data. For instance, as highlighted above, each molecular probe is location-specific, meaning that it contains an oligonucleotide sequence position code204dthat is unique to a specific location of a biological sample. By way of the present techniques, the molecular probes are then delivered inside the cells at specific locations of the biological sample corresponding to the oligonucleotide sequence position code204deach of molecular probe carries. As shown in step414, the cDNA sequence204′/molecular probes can be amplified by PCR.

As described in conjunction with the description of step104of methodology100above, the molecular probes with unique oligonucleotide sequences are then linked to a vessel such as a retrovirus, coupled to disulfide-linked cell-penetrating peptide (CPP) or bead micro-particle which will permit transfer of molecular probes into the cells at specific locations of the biological sample. For live cells, retroviruses such as lentiviruses like the MMLV can be employed as the vessel. SeeFIG.5. Lentiviruses such as MMLV are advantageous as gene delivery vehicles because they are able to stably integrate into the genome of cells. Further, among retroviruses, lentiviruses have the distinguishing property of being able to insert genetic material into both dividing and non-dividing cells. The process for modifying retroviruses such as lentiviruses for use as vectors for gene delivery into cells is well known to those of ordinary skill in the art.

A disulfide-linked cell-penetrating peptide (CPP) or activatable cell-penetrating peptide (ACCP) is also a suitable vessel for transferring the molecular probes into the cells of the biological sample when the sample is live cells or tissue containing live cells. SeeFIG.6. CPP are biocarriers that are able to penetrate biological membranes and thus translocate into cells, thereby permitting the cells to internalize different cargo molecules. According to an exemplary embodiment, the CPPs are short polycations attached via protease-cleavable linkers to neutralizing polyanions. Thus, as shown inFIG.6, the disulfide-linked CPP-to-oligonucleotides complexes are non-permanent in the reducing environment within the cells. As such, once the CPP biocarriers deliver the molecular probes into the individual cells of the biological sample, the disulfide bond between the CPP biocarrier molecule and the molecular probe can be cleaved. For a general description of CPP molecules as biocarriers see, for example, Gagat et al., “Cell-penetrating peptides and their utility in genome function modifications (Review),” International Journal of Molecular Medicine 40: 1615-1623 (October 2017), the contents of which are incorporated by reference as if fully set forth herein.

For tissue with living cells, bead micro-particles702are also a suitable vessel for transferring the molecular probes into the cells of the biological sample. SeeFIG.7. Beads micro-particles702are able to permeate the biological membranes of cells by a process called bead transfection. For instance, micro-particle beads such as glass beads can first be incubated in a solution containing the molecular probes. The micro-particle beads now conjugated with molecular probes can then be introduced into the cells using a process such as electroporation. According to an exemplary embodiment, the bead micro-particles702are the same as bead202described above (seeFIG.2). Other vessel delivery mechanisms (such as a retrovirus, coupled to a disulfide-linked CPP, etc.) are needed because some mechanisms are better than others, depending on if they are being used for tissues or cells.

As described in conjunction with the description of step106of methodology100above, a liquid cargo delivery device such as a microfluidic probe is employed to deliver the vessels with unique molecular probes to specific locations of the biological sample, where the vessel delivers the location-specific molecular probes inside the cells at those locations. See, for example,FIG.8. In step810, a liquid cargo delivery device802(such as a microfluidic probe) scans the surface of a biological sample804and deposits the vessels with unique molecular probes at specific locations (x,y) in the biological sample804. A microfluidic probe is a non-contact, scanning platform that can hydrodynamically localize as little as 100 picoliters of a liquid cargo with micrometer precision.

By way of example only, the liquid cargo delivery device802dispenses a controlled amount of a processing solution (e.g., an aqueous solution) containing the vessels/molecular probes at multiple locations (i.e., (x1,y1), (x1,y2), (x1,y3), etc.) in the biological sample804. See step812. As provided above, once the vessel/molecular probe is delivered to a specific location of the biological sample804, the vessel delivers the molecular probes inside the cell(s)806at that specific location.

After the location-specific molecular probes have been delivered/inserted into the cells806, the cells806are extracted from the biological sample804. See step814. However, even after being disassociated from the biological sample804, the individual cells806retain the molecular probe with oligonucleotide sequence encoding the original position of the cells806in the biological sample804. Thus, this positional data can be retained through the subsequent sequencing process. See step816.

For example, one or more single cell sequencing techniques can be performed. Suitable single cell sequencing techniques include, but are not limited to, drop-seq, seq-well, cyto-seq, and combinations thereof. The single cell sequencing performed in step816can be used to identify the subject cell by the cell barcode (see above), the original position of the cells806within the biological sample804via the unique, location-specific oligonucleotide sequence of the molecular probes, and/or transcriptome information of the cells806. Therefore, the combination of the present positional delivery and encoding process with extraction and single cell sequencing can collect concomitant spatial and molecular measurements (e.g., position coordinates and transcriptomes of one or more of the cells806in the biological sample804) which, as described in conjunction with the description of step110of methodology100above, can be recorded and/or analyzed in silico.

Turning now toFIG.9, a block diagram is shown of an apparatus900for implementing one or more of the methodologies presented herein. By way of example only, apparatus900can be configured to implement one or more of the steps of methodology100ofFIG.1. For instance, according to an exemplary embodiment, apparatus900may be configured to store and/or analyze the transcriptomic data extracted from the single cell sequencing along with the unique positional data obtained from the molecular probes indicating the original positioning of the cells in the biological sample.

Apparatus900includes a computer system910and removable media950. Computer system910includes a processor device920, a network interface925, a memory930, a media interface935and an optional display940. Network interface925allows computer system910to connect to a network, while media interface935allows computer system910to interact with media, such as a hard drive or removable media950.

Processor device920can be configured to implement the methods, steps, and functions disclosed herein. The memory930could be distributed or local and the processor device920could be distributed or singular. The memory930could be implemented as an electrical, magnetic or optical memory, or any combination of these or other types of storage devices. Moreover, the term “memory” should be construed broadly enough to encompass any information able to be read from, or written to, an address in the addressable space accessed by processor device920. With this definition, information on a network, accessible through network interface925, is still within memory930because the processor device920can retrieve the information from the network. It should be noted that each distributed processor that makes up processor device920generally contains its own addressable memory space. It should also be noted that some or all of computer system910can be incorporated into an application-specific or general-use integrated circuit.

Optional display940is any type of display suitable for interacting with a human user of apparatus900. Generally, display940is a computer monitor or other similar display.

Characteristics are as follows:

Service Models are as follows:

Deployment Models are as follows: