CRISPR-Cas gene editing has revolutionized experimental molecular biology over the past decade and holds great promise for the treatment of human genetic diseases. Here we review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, focusing on the assessment and improvement of their editing precision and safety, pushing the limit of editing specificity and efficiency. We summarize the capabilities and limitations of each CRISPR tool from DNA editing to RNA editing, and highlight the opportunities for future improvements and applications in basic research, as well as the therapeutic and clinical considerations for their use in patients. CRISPR-Cas tools have shown exceptional promise in genome engineering over the past decade. Here the authors review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, as well as their editing precision, off-target effects, and clinical considerations. In 2012, the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) was engineered for RNA-programmable DNA cutting3. Since then, CRISPR-Cas systems have been used to manipulate the genomes of cultured and primary cells7, animals8 and plants9, driving major advances in the life sciences and being tested in multiple clinical trials12. In light of the increasing number of new CRISPR-Cas types13, we focus on CRISPR–Cas9/Cas12 in this Review as they are the most widely used genome editors.Genome editing, which involves the precise and efficient manipulation of DNA sequences within the human genome to alter cell fates and organism traits, has the potential to cure genetic diseases14. In contrast, base editors and prime editors precisely install targeted point mutations without requiring DSBs or donor DNA templates15.CRISPR-Cas DNA editing induces double-strand breaks (DSBs) that are repaired predominantly by non-homologous end-joining pathway (NHEJ) and homology-directed repair (HDR). While NHEJ results in small insertions or deletions (indels) and gene disruption at the target sites, homology-directed repair (HDR) can result in the incorporation of a sequence from the exogenous DNA template at the DSB site, though the efficiency may be limited and is inactive in quiescent cells17. Briefly, this system uses a combination of several components to install desired cargos (up to 10 kb) at target loci, including the transposase operon , donor transposase DNA substrate (LE-cargo-RE), and CRISPR-based DNA-binding and R-loop-generating proteins . Engineered Cas9-fused integrase/recombinase, which can in theory insert, delete, invert or replace target DNA, have also been reported to modify genomic DNA in mammalian cells, albeit to date with low efficiency and substantial target sequence restrictions19.CRISPR-associated transposases, Tn6677 and CAST, allow for integration of larger DNA cargos but so far have shown activity only in bacterial cells20. Unlike genome editing, which is permanent, the effects of RNA editing are transient and reversible. Precise RNA editing may also be achieved by recruiting endogenous ADARs with engineered ADAR-recruiting RNAs, without the need to introduce foreign editing enzymes or exogenous ADARs protein into edited cells22. Therefore, RNA base editing has therapeutic potential as a complementary approach to genome editing.In addition to DNA editing, adenosine deaminases acting on RNA (ADAR) fused to dCas13 have been exploited to achieve precise adenosine-to-inosine editing of target RNAIn this review, we cover recent advances in the development and application of CRISPR-based genome editing and RNA editing tools, including CRISPR–Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, focusing on the improvement of their editing efficiency and specificity. We also summarize various NGS-based methods that have been developed to explore unintended genetic alterations and evaluate the safety concerns during gene editing. Finally, we provide perspectives on future directions as well as therapeutic and clinical considerations in this rapidly progressing and exciting field.15.Class 2 CRISPR-Cas systems have been classified into three types, that is type II, type V and type VI, with their corresponding effector proteins Cas9, Cas12, and Cas13, respectively. Among them, most Cas9 and Cas12 variants possess DNA endonuclease activity, while Cas13 variants show preferential RNA-targeting and cleavage activity3. Mechanistically, following R-loop formation by the Cas9-guide RNA ribonucleoprotein complex, the HNH nuclease domain cleaves the guide RNA-bound target DNA strand, while the RuvC-like nuclease domain cleaves the PAM-containing non-target DNA strand 3 bp upstream of the PAM24 , which were engineered by fusing the CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) into a single RNA molecule, predominantly making a blunt-ended double-strand break (DSB) within the protospacer sequenceM24 Fig. . Cas9 cae28 Fig. .Fig. 1Ov30, raising safety concerns for their use in clinical applications and urgent needs to systematically identify these off-target events. Briefly, methods for the genome-wide identification and characterization of potential CRISPR-Cas nuclease off-target sites fall into three general categories: in silico bioinformatic prediction based on sequence homology, in vitro identification of cleavage sites using genomic DNA templates, and in cellulo capturing of off-target editing events31  that nearly all of the sequencing reads are background reads (not associated with CRISPR-Cas induced DSBs). CIRCLE-seq utilizes an alternate genomic substrate, DNA circles, for the in vitro genome-wide identification of nuclease off-targets41. Mechanistically, genomic DNA is sheared into linear fragments, circularized by intramolecular ligation, and incubated with Cas nuclease during which the circularized DNA molecules containing off-target site are linearized. These linearized fragments are then selectively amplified and sequenced to identify the off-targets41 43  complex. Digenome-seq is a commonly used technique based on in vitro Cas nuclease-digested whole-genome sequencing (WGS)While in vitro cell-free biochemical methods are rapid and conventional, they inevitably capture some pseudo off-target sites compared to the in cellulo methods which require the introduction of Cas nuclease directly into cells to identify genome-wide off-target sites. These unbiased, NGS-based methods fall broadly into four categories: indirect labeling of off-target breaks by repaired products , indirect labeling of off-target breaks by DNA repair protein (DISCOVER-seq), direct labeling of unrepaired break ends in situ , and indirect labeling of off-target sites by capturing R-loop mediated ssDNA (CROss-seq) Table .44. Following dsODNs insertion, the specific sequence and adjacent genomic sequences are selectively amplified and sequenced  and the DNA donor (biotin-iGUIDE donor) to increase the off-target capturing rate, as well as UMI incorporation via Tn5 tagmentation to avoid PCR bias46. The limitations of GUIDE-seq based methods include the requirement for high transfection efficiency of the dsODNs into cells, and limited use for blunt-ended DSBs. HTGTS was initially developed as a powerful technique that can generate a genome-wide chromosomal translocations map from a fixed DSB “bait” induced by either I-SceI47 or CRISPR–Cas948. HTGTS utilizes chromosomal translocations between Cas nuclease-induced on-target DSB (“bait”) and off-target DSBs (“prey”) to capture genome-wide off-target sites  which is especially useful for pre-clinical and clinical evaluations. Although HTGTS-based methods are powerful with high sensitivity, the limitation is Cas nuclease-induced translocations are inherently rare events that require large numbers of input genomes for efficient detection. IDLV capture55 and ITR-Seq56 use integrase-defective lentiviral vector and adeno-associated viral (AAV) vector to identify off-targets, respectively; while PolyA-seq detects off-targets by capturing de novo LINE-1 retrotransposon insertions at CRISPR-Cas induced DSBs57  into CRISPR-Cas off-target sitesced Fig. . Using ates Fig. . LAM-HTG60 92, plasmids94 and small DNA fragments95. Recently, we reported that LINE-1 retrotransposons can occur at CRISPR–Cas9 editing sites, in a reverse transcriptase (RT) activity-dependent and endonuclease (EN) consensus motif-independent manner57. These events exploit the availability of frequent DSBs generated by CRISPR–Cas9 nuclease to favor the insertion of exogenous DNA fragments using microhomology-mediated end joining (MMEJ)96. Although chromosomal rearrangements are likely rare events, the concern is that even a very small number of CRISPR–Cas9-edited cells harboring these detrimental events may clonally expand in vivo97 and cause diseases.Though genome editing with CRISPR–Cas9 holds great promise for the treatment of human genetic diseases, it results in a mix of intended and unintended genetic alterations. More rarely, DSBs induced by CRISPR–Cas9 can lead to larger genomic rearrangements including large chromosomal deletions, inversions or translocationsThe way to reduce off-target effects is to optimize the different components of the CRISPR-Cas systems. The selection of sgRNAs that yield high editing efficiency balanced with low off-target activity is likely one preferred strategy for increased safety, especially for clinical applications. In addition, several modifications have been developed for reducing CRISPR nuclease-induced off-target effects and genomic rearrangements while retaining robust on-target activity, including sgRNA modifications, Cas9 protein optimizations, Cas9 system modifications, and delivery improvements.98, chemically modified sgRNAs 99, light-induced degradable sgRNA100, and incorporated bridged nucleic acids  or locked nucleic acids (LNA) at specific locations in crRNAs101 have been shown to broadly reduce off-target activity of CRISPR–Cas9 by several orders of magnitude. Interestingly, since short gRNAs (less than 16 nt in length) effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity, CRISPR GUARD was developed as a method for protecting off-targets sites by co-delivery of short gRNAs to compete with the on-target sgRNA102, similar to co-administration of dead-RNAs (dRNAs)103.Truncated sgRNAs (tru-gRNAs) (with only 17 or 18 nucleotides)104, FeCas9 51, and SpCas9-HF1 105 attenuate contacts with the phosphodiester backbone to be trapped in an inactive state when bound to mismatched targets, while HypaCas9 106 increase the stringency of the allosteric regulation of HNH domain activity with high genome-wide specificity. HiFi Cas9 (R691A) demonstrates superior on-target editing with RNP delivery compared to eSpCas9 and SpCas9-HF1, with decreased off-target activity107. In addition, evoCas9 108, Sniper Cas9 109, xCas9 110, Cas9_R63A/Q768A111 and SuperFi-Cas930 were reported to have lower off-target activity.Besides sgRNA modifications, high-fidelity SpCas9 proteins are generated and further optimized. eSpCas9 115. The Wolfe laboratory developed a Cas9-pDBD (programmable DNA-binding domain) system that restricts its ability to engage its target site, increasing specificity up to 160-fold at off-target sites116. Fusing a minimal motif to spCas9, MiCas9 increases large-size gene knock-in rates and reduces undesirable on-target and off-target indels by the enrichment of RAD51 at target sites117. Small indels and large deletion frequency are shown to be decreased in cells deficient for the central resection gene Nbn and the microhomology-mediated end-joining gene Polq81. The Hu laboratory reported the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites118. Thus, Cas9TX is generated by fusing Cas9 with optimized TREX2, an exo-endonuclease that prevents perfect DNA repair and thereby avoids repeated cleavage, eliminating chromosomal translocations during genome editing both in cell models118 and a mouse model of age-related macular degeneration119. More recently, they showed that compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of off-target effects, chromosomal translocations, large deletions, and plasmid insertions80.A SpCas9 nickase cleaves only one DNA strand by mutating one of the nuclease domains (D10A or H840A). Double nicking by paired Cas9 nickases showed greatly enhanced genome editing specificity2. RNPs have been shown to reduce off-target effects as they act on-target DNA immediately after transfection and are rapidly degraded by proteases and ribonucleases in cells122. For example, exosome-mediated delivery of Cas9 RNPs have been shown for tissue-specific gene therapy of liver diseases123. Moreover, considering the numerous plasmid DNA or viral DNA integrations at editing sites, RNPs could be a safer delivery method for CRISPR-Cas tools. Though the endogenous LINE-1 retrotransposons may still be a potential threat during RNPs-mediated CRISPR-Cas editing, base editors or prime editors could be used as a safer alternative consistent with their reduced DSBs57.CRISPR-Cas can be delivered into cells either as plasmid DNA or mRNA encoding their expression, or directly as proteins or ribonucleoproteins (RNPs)15. Two classes of DNA base editors, cytosine base editors (CBEs) and adenine base editors (ABEs), have been developed to precisely install targeted point mutations without requiring DSBs or donor DNA templates14. In CBEs, the cytidine deaminase APOBEC/AID is fused with nickase Cas9 for targeting via sgRNA and with UGI to increase the accuracy and efficacy of base editing, converting C•G base pairs to T•A base pairs125. In ABEs, a laboratory-evolved TadA deaminase is fused with Cas9 nickase to convert A•T base pairs to G•C base pairs126. Similarly, C-to-G base editors (CGBEs)  consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung), converting C•G base pairs to G•C base pairs, expanding the editing scope of base editors  resulted in the most advanced and efficient AncBE4max or ABEmax134, though this increased activity may also increase the off-target effects. ABEmax was optimized from ABE7.10 and consists of a wild-type TadA monomer and an evolved TadA monomer, while the wild-type TadA monomer has been shown not required for ABE activity136. Thus, ABE8e was developed with only one phage-assisted further evolved TadA monomer, showing the highest efficiency136  to specifically nick the non-edited strand to increase the editing efficiency136 Fig. .137, SPACE138, Target-ACEmax139, STEMEs140, and ACBE141) have been created by fusing cytidine deaminase and adenine deaminase together with Cas9 nickase to concurrently achieve C-to-T and A-to-G conversions at the same target site. More recently, the Lai laboratory fused a CGBE with ABE to develop a new type of dual-deaminase-mediated base editing system, called AGBE system, that can simultaneously introduce 4 types of base conversions  as well as indels and thus can be used for saturated mutagenesis142  , the Yang laboratory showed that in mouse embryos, CBEs generate genome-wide substantial off-target single nucleotide variants, which are enriched in the transcribed regions of the genome143, similar to another excellent work in rice144. These findings were further confirmed and expanded by Digenome-seq146, EndoV-seq147, Orthogonal R-loop assay149, and CROss-seq64, showing ABEs could also generate genome-wide off-targets at a much lower frequency.Though DNA base editors may be more precise tools compared with CRISPR-Cas nuclease152, consistent with the RNA editing ability of deaminase APOBEC1 or TadA.Besides DNA off-targeting, transcriptome-wide RNA-seq was also used to detect off-targets at the RNA level. Three independent groups demonstrated that both CBEs and ABEs can cause substantial (tens of thousands of) off-target RNA SNVs in human cells124. Deoxyuridine (dU) capturing has been well established by NGS-based high-throughput methods such as Excision-seq153, dU-seq154, UPD-Seq155, U-DNA-Seq156 and AI-seq157. By capturing the base editing intermediate dU, a more advanced technique Detect-seq158 is able to trace both the on-target and off-target editing events of CBE, as recently validated by a similar technique called Ucaps-seq159. Mechanistically, Detect-seq uses an in vitro-reconstituted base-excision repair (BER) reaction to achieve specific labeling of dU, during which the normal dTTP or dCTP are replaced by biotin-dUTP or 5-formyl-deoxycytidine triphosphate (5fdCTP) to allow biotin pulldown of the dU-containing DNA or tandem C-to-T transitions , respectively158. In addition to Cas9-dependent and Cas9-independent off-targeting, Detect-seq discovered CBE edits outside the protospacer sequence (out-of-protospacer editing) and on the non-edited strand (target-strand editing), expanding the knowledge that CBEs could induce proximal off-target mutations158.CBEs catalyze C-to-dU conversions and finally result in C-to-T transitions160. Intriguingly, using the same technique, Detect-seq found that DdCBEs could induce extensive off-target editing in the nuclear genome in a TALE array sequence (TAS)-dependent or TAS-independent manner161. Importantly, the authors engineered DdCBEs in three different ways to alleviate such nuclear off-target effects.DddA-derived mitochondrial base editors (DdCBEs), which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins, enable targeted C-to-T conversions specifically in mitochondrial DNAStrategies to reduce off-target effects of DNA base editors are similar to CRISPR-Cas nuclease, including modified guide RNAs, engineered Cas9, delivery improvements, and engineered high-fidelity cytidine or adenosine deaminase.163. A Cas-embedding strategy, which introduces deaminase (APOBEC1 or TadA) into the middle of Cas9n at tolerant sites instead of the N-terminus of nCas9, significantly reduces gRNA-independent off-target editing by DNA base editors164. In contrast to plasmid DNA/viral delivery, delivery of the BE systems via RNP complex or mRNA/sgRNA or lipid nanoparticles (LNPs) result in more transient editing activity and hence improved specificity with fewer off-target effects145.High-fidelity Cas9 variants, such as eSpCas9, SpCas9-HF, HypaCas9, and SuperFi-Cas9 in the form of nickase, were incorporated into BE architecture to decrease Cas-dependent off-target effects165. In CBEs, these variants include SECURE-BE3150, eA3A-BE3166, hA3G-CBE167, AALN-BE4148, YE1-BE4148, transformer BE (tBE)168, AID-2S/AID-3S169, and other next-generation CBEs171. In ABEs, SECURE-ABE135, ABE7.10-F148A152, ABEmaxAW or ABEmaxQW151, ABE8e-V106W136, ABE-del153172 and ABE9 (N108Q/L145T)173 were created to reduce RNA off-targeting and narrow the editing window. Of note, many of these mutations may reduce the deaminase activity. Thus, increased specificity could be a tradeoff for reduced efficiency in selective therapeutic contexts.By engineering cytidine or adenosine deaminase, several high-fidelity BE variants have been reported to minimize the DNA and RNA off-targeting175. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination, representing the first unnatural cytosine deaminases besides the natural AID/APOBEC protein family. Briefly, Td-CGBE performs highly efficient and precise C-to-G editing175, while Td-CBEs175 and TadCBEs174 generate precise C-to-T base conversions with the fusion of UGI and additional mutations. Moreover, TadA dual-base editor (TadDE) was shown to perform equally efficient cytosine and adenine base editing concurrently (C-to-T and A-to-G)174, providing a highly efficient and small size alternative besides the previously reported dual editors that fuse both cytidine and adenosine deaminases (ACBEs)141, as well as enabling saturation mutagenesis and screening.Since ABEs show lower DNA and RNA off-target activity, smaller size and higher on-target editing efficiency than CBEs, two groups re-engineered ABEs for efficient and specific CRISPR-based cytosine base editing178.Prime editors (PE) enable precise genome editing, including targeted small insertions, small deletions, and all 12 possible types of point mutations and their combinations without requiring DSBs or donor DNA templates. PEs use Cas9 nickase (H840A) fused to an engineered Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) that is programmed with desired edit-containing pegRNA. The pegRNA contains the sgRNA, a primer binding site (PBS) and a reverse RNA template (RTT) for a reverse transcriptase to generate the desired edit at the target site176 201, GRAND editing202, Bi-PE203, and PETI204, as well as a similar technique PEDAR that fuses Cas9 nuclease to reverse transcriptase (PE-Cas9) and combining it with two pegRNAs205. Besides highly efficient replacement and large deletions, twinPE enabled precise large insertions  or large inversions (40 kb) in human cells when combined with a site-specific serine recombinase, expanding the capabilities of gene editing for the correction of large or complex human pathogenic alleles201, similar with PASTE which uses a CRISPR–Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads (up to 36 kb)206.Instead of single-pegRNA-mediated PE system, several groups independently claimed that two pegRNAs encoding the same edits in both sense and antisense DNA strands enable programmable replacement or large deletions with high editing efficiency Fig. . This ex176. Likewise, using two pegRNAs could also create DSBs; thus, their off-target and unintended on-target effects need to be carefully evaluated. Additionally, several studies have shown that reverse transcription of 3’-extended pegRNAs (RTT-PBS sequence) can proceed into the tracrRNA scaffold, resulting in massive scaffold sequence insertions at the prime editing site with various insertion lengths207. Therefore, splitting the pegRNA into sgRNA and a separate RTT-PBS sequence (either in a linear or circular form) not only prevent 3’-extended pegRNAs degradation by nucleases185, but also may be used to reduce the pegRNA scaffold insertions.Owing to the potential DSBs created by two nicks in PE3, there is a certain level of genomic fragments, plasmid and LINE-1 insertions observed at on-target sites edited by PE3, though at much lower frequency than CRISPR–Cas9 nuclease; while PE2 or PE3b editing has rare insertions consistent with their reduced DSB formation208, CROss-seq64, and TAPE-seq59 were used to screen for genome-wide Cas-dependent off-target sites of PE systems, demonstrating that PEs provide highly specific genome editing with very limited off-targets which could be further improved by high-fidelity Cas9 variants208. On the other hand, three recent studies showed PE3 does not induce significant Cas-independent off-target mutations/rearrangements in plants209 or human cells211 both in genomic and transcriptomic level, suggesting high editing specificity of its reverse transcriptase moiety.Meanwhile, the Cas-dependent and Cas-independent off-target effects of prime editors have been investigated. Nickase-based Digenome-seq (nDigenome-seq)212. As an RNA-guided RNA endonuclease (RNase), Cas13 consists of two HEPN domains that together form a composite RNase active center responsible for catalyzing RNA cleavage after target RNA binding214. Cas13a, Cas13b, Cas13d-mediated RNA degradation and gene knockdown exhibit high efficiency and specificity relative to RNA interference, enabling potential therapeutic treatments including nervous diseases and pathogenic viruses217.The class 2 type VI systems with the single effector protein Cas13 are the only CRISPR-Cas systems known to exclusively target RNA, showing promise as tools for knockdown, detection and RNA editing219 222. On the other hand, collateral degradation of bystander RNAs by CRISPR-Cas13 system has limited its applications in vivo. Hence high-fidelity Cas13 variants with minimal collateral effects have been developed, which could further elevate their utility for targeted degradation of RNAs in basic research and therapeutic applications223.Interestingly, activated Cas13-crRNA complexes cleave both target RNA (cis-cleavage) and non-target collateral RNAs (trans-cleavage)219 Fig. , making 224 and RESCUE (RNA Editing for Specific C-to-U Exchange)225 that can be used to introduce A-to-I or C-to-U RNA editing, respectively. To minimize the substantial off-target RNA editing of REPAIRv1, the Zhang laboratory identified a new variant dCas13b-ADAR2DD (E488Q/T375G), termed as REPAIRv2, enabling precise, efficient, and highly specific RNA base editing224.Similar to DNA base editors, catalytically inactivated Cas13 protein (dCas13) has been fused to ADAR2 to develop RNA base editors, such as REPAIR (RNA Editing for Programmable A to I Replacement)21 and LEAPER (leveraging endogenous ADAR for programmable editing of RNA)22, have been developed to circumvent problems caused by ectopic expression of editing enzymes or ADARs protein. To improve the editing efficiency and reduce bystander off-target editing, CLUSTER226, cadRNAs227 and LEAPER 2.0228 were generated as the most advanced RNA base editors by using in silico-optimized CLUSTER guide RNAs226 or covalently closed circular arRNAs228, as well as by using CMV promoter instead of U6 promotor and the depletion of pairings of uridines with off-target adenosines228.More recently, by recruiting endogenous ADARs with different types of engineered ADAR-recruiting RNAs (arRNAs), next generation of RNA base editors, that is RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing)In this section we review the factors that are critical for the use of gene editing techniques to treat human diseases, including therapeutic benefits, efficiency and delivery in vivo, precision and specificity.229. The therapeutic fraction may be estimated from clinical observations for diseases in which somatic mosaicism or chimeric transplantation outcomes have been described231; in other cases, this threshold may only emerge after therapeutic gene editing studies have been conducted. In addition, targeting a sub-threshold fraction of cells may provide an intermediate clinical benefit and some genetic modifications may partially ameliorate a disease, such as by converting a null to a hypomorphic allele232.With regard to benefits, in general these could be considerable for gene therapies that rectify the underlying molecular lesion for genetic disease or target fundamental pathophysiologic features of acquired disease. The timing and therapeutic fraction of target cells to reverse the pathophysiology may vary depending on disease. For disease settings in which irreversible organ damage accumulates with time, providing gene therapy early in the disease course may be advantageous120. For in vivo therapies, the most common delivery is by AAV233, although nanoparticles234 and bare RNPs1 may also be applied. Given that pulse delivery of gene editors to primary cells may result in a modest and short exposure, possible genomic effects as observed in vitro may overestimate possible editing effects. Some genomic repair unintended effects, like micronuclei formation or loss of heterozygosity235, are restricted based on cell cycle stage, so targeting non-dividing cells could possibly avoid these effects (as compared to targeting dividing cells during in vitro culture). Other genomic effects may depend on the cell state; for example, the frequency of retrotransposition effects may be related to the underlying cellular activity of retrotransposons57. Of note, therapies that target mRNA rather than DNA may require durable delivery or repeated administration.For efficient editing, adequate delivery systems are needed to achieve enough gene modifications  in the required fraction of cells. The efficiency depends not only on the intrinsic activity of the gene editor but also on its delivery method to cells. For ex vivo therapies - such as targeting hematopoietic cells - often the delivery is by pulse of RNP or mRNA/gRNA via electroporation236. For NHEJ repair targeting gene regulatory elements, long deletions could remove coding sequences and lead to loss-of-function alleles74; for other applications in-frame indels could give neomorphic effects. Likewise for base editing, bystander edits or unintended substitutions (like C-to-G for a C-to-T base editor) could produce additional phenotypes, such as due to unintended missense or nonsense alleles in addition to therapeutic modifications.Related to efficiency is precision, which enumerates how many modifications are perfect and how many are undesirable. For intended HDR repair, there might be concurrent NHEJ repair which will be produced in a fraction of cells. For example, accompanying HDR repair of the sickle cell disease mutation would be imperfect repair that might convert a sickle cell disease allele to a β-thalassemia allele, which could give a mixed clinical picture of chimeric healthy, sickle cell disease, and β-thalassemia HSCs each producing blood cells237. Thus, a short pulse of high-level expression may give a desirable balance of efficiency and specificity121.Specificity describes the capacity for editors to produce changes elsewhere in the genome besides the intended target. Some of these off-target effects may be predictable and testable by different methods as described above. However, other effects may be unexpected, stochastic or widely distributed and thus could escape attention of targeted assays. Even whole-genome sequencing may be an insensitive assay given that in theory rare clonal events could be clinically meaningful. As the frequency of these events diminishes (ultimately to a single event in many targeted cells), it may become infeasible to detect them without targeted assays. Often there may be a tradeoff between gene editing efficiency, precision and specificity238, but might be not feasible for other non-accessible tissues.Finally, one more challenge for the field is to identify the models to measure efficiency, precision and specificity. It can be difficult to comprehensively test genomic modifications in therapeutically relevant contexts with experimental models because it is expected that non-human cells would possibly have different off-target sequences compared to human cells given the different genomic sequence. Thus, experiments in non-human models may not fully mimic the in vivo conditions relevant to therapeutic delivery. Improved models, such as humanized mice, may allow testing on-target editing efficiency and off-target effects in certain tissues (like hematopoietic cells) but not in others. Thus, close follow-up and correlative molecular analyses from subjects enrolled in clinical trials may be the only way to truly evaluate comprehensive genomic editing outcomes. These analyses may be learned from studies focused on easily accessible tissues (like hematopoietic cells), from precious tissue biopsies when feasible to collect, and possibly from cell-free DNA239, their safety and associated clinical risks are uncertain. Although an increasing bevy of techniques may be employed to catalog on- and off-target genomic effects, a somatic genetic modification on its own may not portend clinical consequences. Cells spontaneously accumulate DNA damage over time and with cell divisions, so a modest one-time increase in acquisition of somatic genetic variation may not be particularly notable. Overall, most genetic variants are expected to be neutral with few variants that may be deleterious to cells being clinically inconsequential. In contrast, the potentially most concerning category would be those variants that cause a gain-of-function that could promote clonal expansion and tumorigenicity, via activating oncogenes or disrupting tumor suppressors. So far, no examples of unintended tumorigenicity of therapeutic gene editing have been reported to our knowledge in clinical studies, suggesting the frequency of these effects may be extremely low.Since gene editing approaches are still in an early phase of clinical development and no CRISPR-based therapy has been approved for use in patients yet241. In addition, it is known that therapies that require preparative conditioning, like the genotoxic alkylating myeloablative chemotherapy that often accompanies hematopoietic stem cell transplant or the lymphodepletion that attends immunotherapies, may have risks that need to be considered in the overall risk profile but are not attributable to gene editing per se. For example, two cases of AML were observed out of 47 patients undergoing a gene therapy approach for sickle cell disease243. Molecular analyses suggest that insertional mutagenesis was not the cause for these malignant events, so narrowly one could conclude that the gene therapy did not cause the malignancies. In fact, lentiviral therapies for monogenic blood disorders have a highly favorable safety profile without genotoxicity events observed over greater than 700 patient-years, despite their obligate delivery of semi-random genomic insertions to target stem cells244. This observation illustrates that tallying genomic modifications does not easily translate into quantifying clinical risk.Finally, the clinical considerations must extend to the entirety of the therapeutic gene editing procedure, including any collateral risk of cellular delivery and host responses which may represent more likely classes of clinical risk. For in vivo approaches this could include unwanted host responses to gene transfer vectors like viruses  or transposons (Sleeping Beauty) that may cause stress responses, such as interferon responses and DNA damage responses246.The past 10 years have witnessed remarkable progress in understanding and manipulation of CRISPR-Cas systems, especially in the field of genome editing and RNA editing. CRISPR–Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors have revolutionized the life sciences, paving the way for advances in basic research and therapeutic applications. Nevertheless, future work is needed to provide insights into more precise, more efficient and safer editing tools. Besides DNA and RNA editing, other applications of CRISPR-based tools include epigenome and gene regulation, functional screening, and diagnostics have been well-summarized in recent reviewsThere are plenty of sensitive and unbiased methods that have been developed to evaluate the off-target concerns during genome editing, pushing the limit of editing specificity and efficiency. Despite progress, the currently available methods to detect off-target activity of CRISPR-Cas systems are still heterogenous, not extensively compared one to another, and optimized only for few experimental systems. Furthermore, a method validated and approved for clinical use is lacking. Therefore, the challenge is to develop a consensus method that can unbiasedly, sensitively, rapidly and cost-effectively detect the CRISPR-Cas off-targets in most cellular system, including in vivo models. This need will grow even further when the therapeutic applications of CRISPR-Cas will become of broader use. Improved methods to detect off-target activity will guide the efforts for the development of high-fidelity Cas nuclease or cytidine/adenine deaminases with minimized Cas-dependent or Cas-independent off-target editing, shedding light on the future direction of precision medicine. Of note, these minimized off-target effects focus on not only DNA level but also the RNA level, especially for the DNA base editors that may cause substantial RNA off-targeting.228. Nonetheless, future work on specificity and efficiency are still needed to improve the RNA base editing, as well as more advanced RNA base editors or “RNA prime editors” that could modify the RNA in a broader way to further illuminate RNA biology. In addition, other types of RNA editing tools are still need to be developed for specific and safe RNA editing applications, like recently reported RESTART247 which relies on guide snoRNA to revert PTC-induced translation termination. On the other hand, “endogenous DNA base or prime editors” that without the need of foreign Cas nucleases or exogenous deaminase proteins would be an exciting breakthrough for next-generation genome editing.Unlike DNA editing, RNA editing offers an alternative to genome editing in certain therapeutic applications in a reversible and tunable manner, especially for the RNA base editors that do not need to introduce foreign Cas nuclease or exogenous ADARs protein into edited cells. Over-expression of Cas or ADARs proteins is associated with several safety concerns, including substantial genome-wide and/or transcriptome-wide off-target, immunogenicity, oncogenicity, and delivery hurdlesFinally, for any of these considerations to be relevant a therapy must be available to patients. Given that many patients with the genetic disease may have rare or ultra-rare mutations, it may be extremely challenging to develop a personalized therapy. The current regulatory path raises a substantial barrier in terms of the requirement for extensive pre-clinical studies and high cost of goods, which may be of uncertain prognostic importance. Perhaps new regulatory paths could catalyze the development of additional gene therapies, in particular for rare indications, that could maintain a focus on safety while enabling patient access to innovative and complex therapies. Once a therapy enters clinical trials, typically patients must meet strict inclusion/exclusion criteria, so the applicability of novel therapies to different types of patients, including those with more severe disease, may be difficult to extrapolate. After a therapy is approved, the high cost and complexity of therapies may represent a challenge to patient access and scalability. All things being equal, cheaper and simpler therapies would be easier for patients to access.


Deficits in social cognition have been reported in people at ultra-high risk (UHR) of psychosis exclusively using socio-cognitive tasks and in adolescent and young adult mixed population.Aim of this study was (1) to assess subjective experience of social cognition in adolescent help-seekers identified through UHR criteria, (2) to explore its significant correlations with psychopathology and functioning in UHR individuals; and (3) to monitor longitudinally its stability after a 24-month follow-up period.Participants , aged 13–18 years, completed the comprehensive assessment of at-risk mental states and the GEOPTE scale of social cognition for psychosis.In comparison with non-UHR/FEP patients, both UHR and FEP adolescents showed significantly higher GEOPTE total scores. After 12 months of follow-up, UHR individuals had a significant decrease in severity on GEOPTE “Social Cognition” subscore. In the UHR group at baseline, GEOPTE scores had significant positive correlations with general psychopathology, positive and negative dimensions. Across the 2-year follow-up period, social cognition subscores specifically showed more stable associations with general psychopathology and negative symptoms.Social cognition deficits are prominent in UHR adolescents and similar in severity to those of FEP patients at baseline. However, these impairments decreased over time, presumably together with delivery of targeted, specialized models for early intervention in psychosis.


The consequences for the COVID-19 pandemic in the newborns of affected mothers remains unknown. Previous clinical experiences with other infections during pregnancy lead to considered pregnant women and their offspring especially vulnerable for SARS-COV-2. That is, the underlying physiopathological changes caused by the infection  could clearly compromise fetal neurodevelopment.To analyze the impact of maternal SARS-COV-2 infection during pregnancy in early neurodevelopment of infants gestated during the COVID-19 pandemic period compared to those gestated immediately prior (2017-2021).212 pregnant women (14% infected) were followed throughout their pregnancy and postpartum, including newborn development. SARS-COV-2 infection was serologically confirmed during pregnancy. The Brazelton Neonatal Assessment Scale (NBAS) was administered at 6 weeks old by a trained neonatologist to evaluate neurological, social and behavioral aspects of newborn’s functioning. Differences in NBAS scores between cases and controls were tested by ANOVAs. All the analysis were adjusted for maternal age, sociodemographic status, anxious-depressive symptomatology, infant’s sex and gestational age at birth and NBAS, and for the period of gestation (previous or during COVID-19 pandemic).NBAS social interactive dimension was significantly decreased in those infants exposed to prenatal SARS-COV-2 , particularly when the infection occurred before the week 20 of gestation. Gestation during COVID-19 pandemic did not alter NBAS subscales.SARS-COV-2 infection during pregnancy seems to be associated with lower NBAS scores on social dimension in 6 weeks old exposed newborns.No significant relationships.


Dear Editor,1 MiRNAs are short noncoding RNAs that bind to the 3’-UTR of target mRNAs to repress protein production. However, the causal link between miRNAs and TOF and the underlying mechanism has not been established.Tetralogy of Fallot (TOF) is the most common complex congenital heart disease. Besides gene mutations and copy number variants, altered protein function induced by post-transcriptional or translational regulation also contributes to the onset of TOF.2 and China.3 Five miRNAs with upregulated expression were identified in both TOF datasets  to human embryonic stem cell-derived cardiomyocytes (hESC-CMs) based on our previous data  mice in which the expression of exogenous mmu-mir-222 was under the control of the β-myosin heavy chain (β-MHC) promoter  and TOF-related genes in the MGI mouse phenotype database. Among the 82 candidates, we verified 47 downregulated genes and 4 upregulated genes using RT-qPCR  Fig. . SimilarFinally, we examined whether mir-222-induced cardiac defects could be reversed by the miRNA maturation enhancer enoxacin or ferroptosis inhibitor Fer-1 in KI/KI mice. Our results revealed that both enoxacin and Fer-1 treatments significantly reduced embryonic heart defects, respectively, including phenotypes of VSD, subpulmonary stenosis, and overriding aorta Fig. . OverallDICER1-AGO2 signaling in the control of cardiac miRNA maturation/dosage and ferroptosis as potential therapeutic targets for the prevention of TOF.In summary, we identified miR-222 as a master regulator of cardiac miRNA maturation/dosage and demonstrated connections among high levels of miR-222, cardiac hypertrophy, ferroptosis in mouse models and fetuses with TOF Fig. . AlthougSupplementary MaterialsSupplemental Excel File ISupplemental Excel File IISupplemental Excel File III


ScRNA-seq is a powerful tool for investigating the cancer microenvironment, but the cost of analysing every scientific scenario is prohibitive. Fortunately, deconvolution of bulk RNA-seq data with scRNA-seq cell atlas reference datasets provides a cheaper strategy. In this study, we validated the feasibility of deciphering the microenvironment of liver cancer through the estimation of cell fractions with Cibersortx and scRNA-seq atlas reference datasets. Five cell types are associated with patient outcomes, showing that deconvolution is a useful method for characterising the tumour microenvironment.Liver cancers give rise to a heavy burden on healthcare worldwide. Understanding the tumour microenvironment (TME) underpins the development of precision therapy. Single-cell RNA sequencing (scRNA-seq) technology has generated high-quality cell atlases of the TME, but its wider application faces enormous costs for various clinical circumstances. Fortunately, a variety of deconvolution algorithms can instead repurpose bulk RNA-seq data, alleviating the need for generating scRNA-seq datasets. In this study, we reviewed major public omics databases for relevance in this study and utilised eight RNA-seqs and one microarray dataset from clinical studies. To decipher the TME of liver cancer, we estimated the fractions of liver cell components by deconvoluting the samples with Cibersortx using three reference scRNA-seq atlases. We also confirmed that Cibersortx can accurately deconvolute cell types/subtypes of interest. Compared with non-tumorous liver, liver cancers showed multiple decreased cell types forming normal liver microarchitecture, as well as elevated cell types involved in fibrogenesis, abnormal angiogenesis, and disturbed immune responses. Survival analysis shows that the fractions of five cell types/subtypes significantly correlated with patient outcomes, indicating potential therapeutic targets. Therefore, deconvolution of bulk RNA-seq data with scRNA-seq atlas references can be a useful tool to help understand the TME. Liver cancer is one of the leading causes of cancer-related mortality worldwide, making up 4.7% of newly diagnosed cases but 8.2% of deaths . HepatocSingle-cell sequencing (scRNA-seq) technology delivers in-depth interrogation of the TME. The analysis of complex cancer tissues at the single-cell level through scRNA-seq has brought insights into the heterogeneity and progression of cancer, as well as escape from immune surveillance, drug resistance, and intercellular communication . Howeverreg) cells correlate with negative outcomes.In this study, we reviewed major omics databases and selected ten studies that compared transcriptomes between HCC/CCA and normal liver. The cell fractions of tumour and non-tumour tissues were estimated with Cibersortx and three scRNA-seq reference atlases. The included studies contain two cohort studies of HCC, allowing us to investigate the clinical implications of TME abnormalities through survival analysis and gene set enrichment analysis (GSEA) . We deteWe searched three public omics databases—the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and the International Cancer Genome Consortium (ICGC)—for studies engaging liver cancers. Inclusion criteria were: (1) histologically identified sample series from human tissues in clinical studies (including cohort studies and case review series); (2) whole transcriptomes by microarray or RNA-seq; (3) with non-tumorous or normal liver tissues as controls (for comparison) or follow-up information ; (4) studies covering two major liver cancers (HCC and CCA) were included and all other studies were excluded, i.e., metastatic liver cancers and non-malignant hyperplasia . Finally, nine curated datasets (eight RNA-seq and one microarray) were selected for this study .Microarray studies with raw data (CEL files) from the GEO database were obtained via R package SCAN.UPC , which pTCGA-LIHC was obtained via UCSCXenaTools  and dataThree single-cell RNA-seq datasets were used in this study: (1) GSE115469 , (2) GSE146409 (TME-Stroma), and (3) GSE156337 (TME-Immune). GSE115469 is a liver subset of the Human Cell Atlas . GSE1464All expression matrices were normalised to 10,000 counts/cell and packed into an H5AD file with authors’ annotation as metadata for subsequent estimations. Both the signature matrix of scRNA-seq datasets and the expression matrix of bulk RNA-seq datasets were transformed into tab-delimited tables. The signature matrices of reference scRNA-seq were built with the Cibersortx protocol for “scRNA-seq” (“S”). Deconvolution was performed with the “Impute Cell Fractions” module. In validation experiments, batch correction was disabled in within-study validation and activated in “S” mode (with single-cell expression matrix as reference) in cross-study validation . In the estimation of real clinical data, batch correction was enabled in “S” mode for RNA-seq datasets and “B” mode (with single-cell expression matrix collapsed into bulk matrix before used as reference) for microarray datasets. All other Cibersortx parameters were the default configurations .+ T cell + CD8+ T cell + Treg cell).Cibersortx fails when the cell type tree of the reference atlas is complicated . In this situation, collapsing some branches of the cell type tree would complete the calculation . In our The accuracy and robustness of deconvolution with Cibersortx were tested in two groups of experiments: intra-study validation and cross-study validation. In intra-study validation experiments, the signature matrix of scRNA-seq and the pseudo-bulk for testing were generated from the same scRNA-seq atlas. The advantage of this method is full coverage of all cell types/subtypes. In cross-study validation experiments, the signature matrix of scRNA-seq and the pseudo-bulk dataset were generated from different atlases.Pseudo-bulk datasets were generated using the random module of NumPy in the following procedure: the expression of the scRNA-seq atlas was separated into two groups, cell type of interest and all the remaining cell type/subtypes collapsed into a single group labelled as “others”; 10% of cells in each group were selected using Choice function of NumPy, then two representative expression vectors  Pearson correlation coefficient (PCC) between predefined proportions . The patient cohort was first ordered based on descending order of estimated cell fractions and then separated into high- and low-level groups. All separation possibilities (from 1:n-1 to n-1:1) were tested with log-rank tests. The one with the lowest P-value in log-rank tests was selected as the optimised separation. If all the P-values were above 0.05, the cohort was equally separated into two groups (median-point separation).Survival lengths were transformed into months and observed events (labelled as “1”) were transformed into “True” . KaplanPathway activities were estimated by PROGENy . GSEA wahttps://github.com/ErasmusMC-Bioinformatics/OIO_Shaoshi, accessed date 27 December 2022). The supplements and pre-processed datasets were shared with Zenodo .For better reproducibility, all the analysis scripts including pre-processing have been shared with GitHub  and three scRNA-seq atlases . These atlases describe more than 20 cell types or subtypes. We first performed intra-study validation experiments to test whether all identified cell types from each atlas could be accurately deconvoluted with Cibersortx. In this group of experiments, pseudo-bulk datasets were generated by the same atlas . In thisAlthough Cibersortx allows “partial deconvolution”, in which the samples may contain cell type/subtypes not present in the reference atlas, we performed a group of cross-study validation experiments to assess this impact. The results of these experiments demonstrate that the PCC between the presets and prediction remain high , Page 19To determine the difference in cell fraction between tumour and non-tumour tissues, we compared seven RNA-seq datasets which provide paired tissues collected from HCC/CCA cohort studies or case review studies. LIRI-JP is an RNA-seq study that includes primary liver cancers, and secondary liver cancers from stomach, colon, prostate, etc., with adjacent non-tumorous liver tissues as controls. GSE119336 is an RNA-seq study comparing CCA and non-tumour liver. The other five RNA-seq studies compare HCC and non-tumour liver tissues. Three of these studies included cases with HBV infection as the risk factor.The three atlases provide more than 20 cell types, allowing a panoramic view of TMEs. All of these cell types can be largely divided into three groups: (1) cell types underpinning the fundamental physiology of livers, e.g., hepatocytes, cholangiocytes; (2) cell types participating in the pathological evolution of cancer formation, e.g., HSCs, LVECts; (3) immune cells. Results of related cell types are described in the following groups.These two cell types constitute the major functional units of livers—liver lobule and bile ducts . As bothHSCs are tissue-specific equivalents of pericytes, and pericytes can be rarely detected in normal livers ,34. FibrLiver sinusoidal endothelial cells (LSECs) form the wall of hepatic sinusoids and participate in immune responses. Vascular smooth muscle cells (vSMCs) are also key components of blood vessels. Tumour liver vascular endothelial cells (LVECts) are variants of normal LVECs . Decreas+ (helper), CD8+ (cytotoxic) T cells, and Treg cells (TME-Immune atlas) [+ T cells rise sharply in CCA while CD8+ T cells elevate moderately in HCC. Finally, Treg cells are rarely detected in normal livers whereas elevations are common in liver cancers. Treg cells have been recognised as a suppressor of tumour immune responses. Liver cancers also show high levels of overall T cells (CD4++CD8++Treg cells).  TCR chains or gamma–delta (γδ) chains  ; CD4+ (he atlas) . We obse cells). A–F.B cells: The Normal Liver atlas identified two subtypes of B cells, mature B cells (antigen inexperienced) and plasma B cells (antigen secreting) . VariatiMacrophages: The Normal Liver atlas contained inflammatory and non-inflammatory macrophages . The TMEDendritic cells: As the quintessential antigen-presenting cell, the dendritic cell (DC) is another hot research interest for the potential of immunotherapy. The TME-Stroma atlas was used to estimate conventional DC1 and DC2 (cDC1 and cDC2) cell types ,36. Decoreg cells can also be observed, suggesting the disordered responses in tumours.In summary, liver cancers show higher levels of overall immune cells, involving both the innate (monocyte–macrophages) and adaptive branches , as well as auxiliary components (dendritic cells). Meanwhile, suppressive components such as TThis group involves two cell types that can proliferate and differentiate. Bi-potent stem cells (from TME-Immune atlas) were named for their potential to differentiate into both hepatocytes and cholangiocytes . ProlifeThese three atlases also identified some immune cell types which exist in the liver with a relatively low abundance. TME-Immune identified a cluster of natural killer (NK) cells (a key component of the innate immune branch) and a cluster of myeloid cells (the liver-resident precursors of monocytes–macrophages) . The NorFinally, we investigated whether cellular alteration affects clinical outcome of HCC through survival analysis. In public repositories, TCGA-LIHC is the highest-cited cohort of a liver cancer study, with well-annotated follow-up information and substantial sample size (370 HCC patients). TCGA-LIHC is a pooled study of five cohorts with mixed risk factors. Available survival analyses include overall survival (OS) and disease-free survival (DFS) [The distributions of estimated cell fractions show two typical shapes, “Sigmoid” or “Exponential” . Using aAmong all the estimated cell types, hepatocytes and bi-potent cells show substantial impacts on patients’ outcomes. The estimated fractions of hepatocytes show a sigmoid-shaped distribution. The optimisation strategy  separates the cohort at a close-to-median point in OS analysis, and at an inflection point for DFS analysis C. High freg cell (negative).GSE14520 is a study that recruited more than 200 HBV-related HCC cases and provides both OS and DFS information ,39. Its Pathway analysis provides useful information for the identification of therapeutic targets. We also conducted a parallel series of analyses by support vector regression. All Signature matrices and scripts have been shared online (see GitHub/Zenodo address).Deconvolution algorithms draw high interest in estimating cell fractions with scRNA-seq atlases. Cibersortx and MuSiC are two state-of-the-art algorithms in benchmarking studies ,41. We aCibersortx estimates cell fractions by quantifying the abundance of signature genes, which warrants careful consideration when interpreting the results. Cell fractions can be defined in diverse ways. In conventional histological studies, cell proportion calculates by volume, cell number, mass, etc., . The preThree scRNA-seq atlases help portray biological events in liver cancers, including angiogenesis, fibrogenesis, immunity, and stem cell transformation. Agents targeting angiogenesis made the first breakthrough in liver cancer chemotherapy . Cancer Liver carcinogenesis has close ties to chronic inflammations, either viral hepatitis or steatohepatitis. A normal liver resides liver-specific pericytes , which can be inflammation-activated. Protracted inflammation leads to stellate cell transformation and fibroblast proliferation . The latreg cells. Treg cells function through the PD-1/PD-L1 pathway, leading to tumour tolerance. A blockade of this communication results in the resurrection of immune responses in a minor group of patients. Further studies show that the therapeutic efficiency of a PD1 inhibitor depends on the interaction between the TME and other immune components  [Liver cancer evolves with the cross-talk between malignant cells and the immune system. In the early phase of cancer initiation, immune cells actively move towards and fight against transformed cells. Long-term engagement finally resulted in the exhaustion of anti-tumour immunity. Some of the immune cells serve the malignant transformation. Such components include well-established tumour-associated macrophages ,50, exhaT cells) ,54.+ cells were proposed as a tumour-initiating component in HCC development [+ cells. Although the interpretation of these findings warrants careful consideration, our study demonstrates that deconvolution can also help understand the mechanism of cancer formation.The cancer stem cell (CSC) hypothesis proposed that a small cell population harbouring embryonic characteristics fuel tumour growth. It is difficult to identify CSCs except by tracing their descendants bearing specific features. Reported biomarkers of liver CSCs include CD133, CD90, epithelial cell adhesion molecule (EpCAM), etc., . EpCAM+ elopment . EpCAM eelopment . In our elopment ,59. Pathelopment . SurvivaIn this study, we deciphered the TME of liver cancer by estimating the cell fractions of a sample given a transcriptome. By estimating more than 20 cell types/subtypes within bulk RNA-seq data using three atlases and Cibersortx, we found disruptions of normal liver architecture, abnormal fibrogenesis and angiogenesis, as well as disturbed immune responses in HCC and CCA. Survival analysis demonstrated that five cell types/subtypes highly correlated with patient outcomes.Deconvolution algorithm and emerging scRNA-seq atlases allow the decomposition of bulk RNA-seq data into cell-type fractions. By linking the cell fractions of samples and clinical follow-up information, we provide an innovative approach for the discovery of potential therapeutic targets. In the future, with the advent of more high-quality scRNA-seq atlases, deconvolution could be a powerful data mining tool for uncovering the intricate nature of the TME of liver cancer and revealing valuable information in the vast amount of available transcriptomic data.


We then review current and future applications of novel PET biomarkers for imaging the steroid receptors, including the estrogen and progesterone receptors, the HER2 receptor, cellular proliferation, and amino acid metabolism.The goals of precision oncology are to provide targeted drug therapy based on each individual’s specific tumor biology, and to enable the prediction and early assessment of treatment response to allow treatment modification when necessary. Thus, precision oncology aims to maximize treatment success while minimizing the side effects of inadequate or suboptimal therapies. Molecular imaging, through noninvasive assessment of clinically relevant tumor biomarkers across the entire disease burden, has the potential to revolutionize clinical oncology, including breast oncology. In this article, we review breast cancer positron emission tomography (PET) imaging biomarkers for providing early response assessment and predicting treatment outcomes. For 2- The role of molecular imaging, particularly positron emission tomography (PET), continues to increase and evolve in the evaluation of breast cancer. While anatomic imaging  for systemic staging) represent the traditional modalities utilized in breast cancer evaluation, molecular imaging offers the unique advantage of providing in vivo biochemical or metabolic information specific to the tumor. Although molecular imaging includes a variety of imaging detector technologies and probes, this review focuses on PET imaging, which is the most rapidly evolving molecular imaging technology with a growing number of novel tracers.PET provides quantitative images of biologic processes at acceptably low patient radiation doses. In addition, among the various molecular imaging technologies, PET offers high sensitivity and relatively better spatial resolution, improving detection and quantification of regional tracer uptake. It also permits considerable flexibility in imaging probe design that enables the ability to target a wide variety of molecular processes of importance to cancer. The rapid translation of PET to clinical oncology in the late 1990’s and early 2000’s was due to advances in imaging technology, including combined PET/CT and widespread commercial production of the glucose analog FDG. Although utilization and potential applications of FDG-PET/CT continue to grow, novel PET tracers also demonstrate great potential to transform clinical breast oncology.New oncologic therapies that target specific biologic pathways continue to transform clinical breast oncology and improve prognoses. These targeted therapies are typically paired with diagnostic tests (companion diagnostics) that can predict and/or monitor treatment response, allowing for tumor-specific treatment optimization, deemed precision medicine. Thus, in addition to the development of new targeted therapies, research efforts have also sought to identify biological markers (or biomarkers) that can identify and quantify the biologic pathway that is targeted for therapy, and thus predict drug efficacy and quantify a pharmacologic effect.Traditional oncologic biomarkers are tissue-based assays. For example, immunohistochemistry (IHC) for expression of various cell receptors (e.g. the estrogen receptor (ER)) has become standard of care in the diagnosis and treatment of breast cancer. However, there are inherent limitations to tissue-based bioassays. The invasiveness of the biopsies limits the frequency and potential sites of sampling, as well as the number of disease sites that can be sampled. With limited sites of disease sampled, particularly in the metastatic setting, heterogeneity of the tumor burden cannot be assessed or incorporated into the treatment plan. Therefore, imaging biomarkers are of great interest to overcome these limitations by offering a non-invasive assessment of the full burden of disease . PET biohexokinase -19-norpregn-4-ene-3,20-dione (FFNP), with high affinity and selectivity for the PR maleimide (FBEM), demonstrated that tracer uptake on PET correlated with HER2 receptor expression by IHC in a xenograft mouse model. Furthermore, following treatment with 17-DMAG, an inhibitor of heat shock protein 90 that decreases HER2 expression, HER2 expression decreased as measured by 18F-FBEM-PET, as well as on pathology [Multiple research groups have also investigated affibody molecules for imaging HER2. The much smaller size of these proteins compared to antibodies allows more rapid blood clearance and higher tumor penetration, thus precluding the need for delayed imaging. Preclinical studies of affibodies for HER2 imaging have utilized a variety of isotopes. Promising preclinical studies of an athology . Early pathology .89Z-trastuzumab-PET and serial FDG-PET/CT provided high positive and negative predictive value for response to HER2-targeted therapy [As is the case for ER-targeted therapy, the combination of HER2 PET imaging to assess the target and other imaging such as FDG-PET/CT to measure early response may by particularly helpful in guiding HER2-targeted therapy. A nice example is seen in the results of the SEPHIR trial, where the combination of pre-therapy  therapy .Increased cellular proliferation is a hallmark of malignancy, including breast cancer, and is clinically relevant to tumor behavior and growth . Markers11C-thymidine-PET to image tumor cell proliferation and treatment response, the short half-life of 11C and its complex metabolism limited its clinical utility [18F-3′-deoxy-3′-fluorothymidine (FLT) has overcome these limitations, providing a more practical approach and currently representing the primary proliferation imaging agent. Cellular uptake of FLT is dependent on the activity of thymidine-kinase-1 (TK-1) which is relatively overexpressed in proliferating, including malignant, cells, but is low or absent in quiescent cells [The most studied and promising approach to imaging proliferation to date is to image the salvage pathway of incorporating thymidine into DNA. While early studies investigated  utility , 128. Thnt cells , 130. FLnt cells , 131.Compared to FDG-PET, FLT demonstrates lower cellular uptake, and has high uptake in the liver and bone marrow , 129. Almax percent change from baseline to the first post-treatment scan between patients with and without PCR [While early studies of FLT in breast cancer showed promise, several more recent studies of FLT-PET in the neoadjuvant setting demonstrate conflicting results. A study of 20 women found that baseline uptake on FLT-PET/CT, as measured by SUV, was significantly related to Ki67 expression. However, neither baseline FLT uptake nor the change in uptake following 1 cycle of neoadjuvant therapy was predictive of treatment response . A recenhout PCR . Most ofmax was significantly associated with post-therapy Ki67 in surgical specimens, the percent change in FLT uptake did not correlate with changes in Ki67. Although these results suggest that FLT-PET/CT performed pre-therapy and following a short exposure to endocrine therapy, or at a single time point following initiation of therapy, could add value in guiding treatment, further studies are needed to understand how best to apply FLT-PET/CT.A recent study by Romine et al. investigated FLT-PET/CT for assessing early response to aromatase inhibitors in patients with ER-positive operable breast cancer. The study included a subset of women who underwent paired FLT-PET/CT scans prior to endocrine therapy and again pre-operatively, with tissue samples also obtained prior to treatment and at surgery. While the post therapy SUV18F-Iso-1 [Preclinical trials also suggest a role for FLT-PET/CT in evaluating early response to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. Ma et al. demonstrated that FLT-PET/CT could immediately and accurately monitor Palbociclib (a CDK4/6 inhibitor) response in a mouse model of triple negative breast cancer . In a si8F-Iso-1 , 141, ma8F-Iso-1 .18F-flurocyclobutane-1-carboxylic acid (18F-fluciclovine) received FDA approval in 2016 for imaging men with biochemical recurrence of prostate cancer [18F-fluciclovine utilizes the same transporters as native amino acids, namely those involved in glutamine transport, but is not metabolized [18F-fluciclovine washes out over time, and images are obtained soon after injection to measure amino acid transport [18F-fluciclovine-PET, though, is not specific for prostate cancer and has been studied as an investigational agent in other malignancies, including breast cancer.Dysregulated metabolism in malignancy extends beyond accelerated glucose metabolism, providing additional opportunities for PET imaging . Accelere cancer . As a noabolized . Consequransport , a disti18F-fluciclovine-PET has been studied in imaging breast cancer. A 2016 study of 37 women with a new diagnosis of locally advanced breast cancer demonstrated the ability of 18F-fluciclovine-PET to image both invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC). In a subset of patients who were also imaged with FDG, those with ILC had greater uptake of 18F-fluciclovine compared to FDG, while those with IDC had lower uptake relative to 18F-fluciclovine [18F-fluciclovine kinetic parameters between ER-positive, HER2-positive, and TNBC subtypes of breast cancer [18F-fluciclovine in breast cancer remains an active area of research.As a measure of amino acid metabolism, iclovine . Similariclovine . These dt cancer . Utilizi11C-glutamine has been developed and studied in animals [18F-4-Fluoroglutamine (18F-Gln), has also been developed and advanced into clinical trials. Several malignancies have been imaged in patients, including several breast cancer subtypes [18F-Gln measures glutamine pool size, an indirect measure of tumor glutaminolysis [18F-Gln, indicative of a small pool size of glutamine in a tumor with inherently high glutamine metabolism. In ER-positive tumors, increased uptake of 18F-Gln was observed, reflecting an increased glutamine pool size in this tumor subtype that does not actively consume glutamine. This may also explain good uptake of compounds that reflect glutamine transport and pool in ER-positive tumors  has also advanced into clinical trials. Imaging PARP inhibitor drug target expression with FTT may enable better patient selection. Variable FTT uptake was seen among patients with and without BRCA mutations, supporting continued study with this tracer as a biomarker [Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors (PARPi) leverage deficiencies associated with homologous repair that are conferred by tumor mutations in BRCA-related genes, and have proven efficacy in breast cancer. However, response to PARPi is variable despite selection for patients based on known genetic mutations . Imagingiomarker . In addiiomarker . An ongoiomarker , 156 mayThe ongoing development and utilization of novel targeted breast cancer drug therapies has transformed clinical breast oncology and improved prognoses. With these new therapies targeting specific biologic pathways comes great potential value for molecular imaging. Molecular imaging offers a noninvasive bioassay to predict and assess early response to treatment across the entire disease burden. This review highlights the most promising PET breast cancer biomarkers to date for early response assessment and for predicting long-term response, including tracers that are currently approved for clinical use, as well as several that are still exploratory, but warrant further study in larger clinical trials.FDG remains the most widely utilized molecular imaging agent in clinical oncology, including for breast cancer. While FDG-PET/CT has an established role in clinical oncology for staging and restaging advanced breast cancer, its potential applications continue to expand. Research studies have clearly demonstrated the value of FDG-PET/CT for providing an early assessment to neoadjuvant therapy , 27, incAlthough FDG is the most established PET tracer in clinical breast oncology, research suggests potential value of multiple novel PET tracers. FES-PET/CT is already approved for defined indications, but research suggests a greater potential role, including for predicting and assessing response to endocrine therapy. Early studies also show promise for FLT, HER2-imaging tracers, and amino acid metabolism tracers. At this point, larger studies, including multicenter trials, are needed to better define appropriate clinical applications, and to provide the foundation for clinical translation. In addition, for some of these newer tracers, such as FLT, prospective studies need to be conducted in more homogeneous tumor populations (ie single subtypes of breast cancer) and in patients on defined treatment regimens, in order to clarify conflicting results that occur among more heterogeneous studies. Finally, standardized methods for PET imaging acquisition and analysis is needed for novel tracers in order to optimize methods for both research and clinical translation. Multiple PET tracers are poised to make an impact in clinical oncology, pending these next steps in investigation.


Chimeric antigen receptors (CAR) T cells (CAR-T) mark a significant step towards producing safe and effective personal anticancer treatments. CAR-T strategies engineers the T cells from the patients to allow specific binding to a tumour-specific antigen. CAR-Ts are a second-wave offensive strategy to clear out remaining chemotherapy-resistant tumour cells. Though showing practical antitumor abilities in multiple haematological malignancies and solid tumour cancers, the issues of antigen escape, tumour infiltration/penetration, and toxicity side effects limit the usage of prolonged CAR-T therapies. However, engineering immunology has exploited human stem cell-based CAR-T therapies and the development of CAR-M (macrophage) therapies to combat the disadvantages of conventional CAR-T therapies. In this review, we will highlight the challenges of CAR-T therapies and combat them with engineering immunology for cancer immunotherapy. Chimeric antigen receptor (CAR) T cells therapies are one of the most exciting developments in recent cancer immunotherapies. CAR-T cells act as engineered immune cells that track down a targeted antigen, then induce apoptosis on the cells bearing the targeted antigen. Collectively, CAR-T stimulated responses are independent from MCH surface receptor induced immune response. Though there are many clinical trials undergoing FDA and clinical approval currently, there are many challenges that CAR-T immunotherapies still face such as antigen escape and cytokine release syndrome, discussed later in this review. Current research are also focusing on implementing CAR constructs on other immune cells such as macrophages and improving the safety profile of current CAR-T cells. In this review, we review the current progress of CAR-T immunotherapy and some recent clinical trials, as well as challenges of CAR-T immunotherapy and alternative solutions.The CAR construct was genetically modified and plated to thousands of patients’ T cells in the laboratory by adding a gene for an artificial CAR protein. This manufacturing retains the specificity of the antibody immune system and enables T cells to identify and latch onto a specific antigen on the malignant cells and destroy them. The CAR comprises three domains, an extracellular antigen recognition domain, a transmembrane spacer domain, and an intracellular domain . Currentin vitro and helped boost CAR T cells’ expansion and antitumor efficacy , of the CAR are derived from the Fab of the monoclonal antibodies, which are the variable light (VL) and heavy (VH) chains . Though efficacy  , there have been studies focused on the effectiveness of CAR-T therapy on a variety of haematological malignancies and solid tumours. This section will discuss CAR-T efficiency in a few cancers that have been proven to be most significant or provided the most significant clinical data.CAR-T immunotherapy has demonstrated remarkable effects in numerous haematological malignancies, including acute lymphoblastic leukaemia (ALL), chronic lymphoblastic leukaemia (CLL), lymphomas, as well as multiple myeloma (MM). In ALL patients, thanks to Kymriah developed by Novartis, the CD19-targeting CAR-T therapy showed significant antitumor effects when initially treated . HoweverUnlike blood cancers, where cancer cells express special or unique surface protein markers, solid tumour cells normally do not express one tumour-specific marker . InsteadIn ovarian cancer, there is a high level of tumour recurrence even after surgery and chemotherapy procedures, leading to an increased demand for alternative solutions. Tumour-associated glycoprotein 72 (TAG72) is highly expressed on the cancer cell surface, leading to research on anti-TAG 72 CAR-T cells . AnotherIn brain cancers, the most common type of primary malignant brain tumors is glioblastoma (GBM). Current available treatment is tumour removal surgery followed by chemotherapy, which yields the 2 years survival rate of patients below 30 percent . CurrentThe use of CAR-T immunotherapies in colorectal cancers are heavily investigated. The carcinoembryonic antigen (CEA) is a common target for current CAR-T immunotherapies as the surface marker expressed on over 80 percent of colorectal cancers . IntraveWith growing interest in producing CAR-T immunotherapy products for the general public, an increase in CAR-T-related clinical trials has been initiated. In this section, we will briefly discuss some clinical trials as examples.In a clinical trial regarding haematological malignancies performed by Though CAR-T immunotherapy has many advantages compared to traditional chemotherapy and surgery, CAR-T technologies are still under heavy advancement due to some challenges it faces. In this section, we will briefly discuss CAR-T immunotherapy’s limitations from the angle of antigen escape, issues with infiltration, CAR-T toxicity challenges, and lastly, some current modifications to improve the CAR-T safety profile.One of the most common observations during the later period of CAR-T immunotherapy treatments is tumours developing resistance to single antigen targeting constructs. The phenomenon, called antigen escape, is seen in later stages of treatment as tumour cells lose particle or complete expression of target antigens. In the case of targeting CD19 in acute lymphoblastic leukaemia, 30–70 percent of patients showed downregulation or loss of target CD19 expression after treatment . SimilarSolid tumours are more effective at using toxic tumour microenvironment and physical barriers to block interaction from immune cells. While tumour facilitating cells, such as myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAMs), and regulatory T cells (Tregs), can migrate into the tumour microenvironment, many immune cells cannot . On top in-vivo T cell expansion, a sign of CAR-T-related toxicity  are essential to bridging the gap between the adaptive and innate immune systems. Recently, Heczley et al. created GD2 ganglioside CAR-NKTs targeting neuroblastoma cells. The authors articulated that the generated anti-GD2 CAR-NKTs are toxic to GD2-expressing neuroblastoma cells as well as PD-1-expressing macrophages. On top of that, the authors found that the CAR-NKT efficiency is highly dependent on costimulatory domains such as, as mentioned previously, CD28 and 4-1BB domains. Unsurprisingly, the constructs with both costimulatory domains exhibit the best antitumour effects and longest persistence. Later, pacities . Though Via artificial modification of CTLA-4 pathways, CTLA-4 blocking cannot single-handedly break TME and aid T cell efficiency significantly, as suggested by Eager’s team (via macrophage-initiated tumour immunities (As mentioned a few times before, immune checkpoints are essential to enhance immune cells’ ability to destroy cancer . The twor’s team . Howeverr’s team . These fr’s team . It perhr’s team . Howevermunities .Improving CAR constructs and its related CAR-T or CAR-M cancer immunotherapies has been an important focus over the last decades. CAR-T therapies have achieved impressive clinical results while the CAR-M field is moving rapidly forward. CAR-T immunotherapy is a very good starting point for the research of future CAR-related therapies. Perhaps when CAR-T therapies are safer and wider in use, they can be used as an initial treatment for naive and early-stage tumours that are easier to target by CAR-Ts or as a last-ditch resort to target as much tumour tissue as possible. Either way, CAR-T therapies, for now, should not be considered a prolonged treatment for patients as there are still many challenges we are facing. The CAR-T and CAR-M fields have complementary effects and disadvantages, further research should focus on minimizing the side effects of each immunotherapy while maximizing the strengths. Other recent reviews on CAR-T therapeutics such as Han et al., 2021 also discuss the therapeutics of CAR-T in haematological malignancies. Their findings are similar to ones presented in this article, an increase in CAR-T treatments in different blood cancers though with many complications. As Sterner also presented, there are many complications regarding CAR-T therapy in general. However, there are potential to expand CAR-T therapy to a wider range of cancers. Looking for alternate or complementing strategies to improve CAR construct efficiencies to replace current traditional cancer therapies such as chemotherapies and invasive surgeries. When effective CAR-mediated immunity is established, we can look forward to integrating with other fields of genomics, transcriptomics, and proteomics to obtain early diagnostics of cancers and effective treatments to avoid prolonged treatments and improve patient satisfaction.


Polymers are well-recognized carriers useful for delivering therapeutic drug and imaging probes to the target specified in the defined pathophysiological site. The functional drug molecules and imaging agents were chemically attached or physically loaded in the carrier polymer matrix via cleavable spacers. Using appropriate targeting moieties, these polymeric carriers (PCs) loaded with functional molecules were designed to realize target-specific delivery at the cellular level. The biodistribution of these carriers can be tracked using imaging agents with suitable imaging techniques. The drug molecules can be released by cleaving the spacers either by endogenous stimuli  at the targeted physiological site or exogenous stimuli . Recently, two-photon absorption (2PA)-mediated drug delivery and imaging has gained significant attention because TPA from near-infrared light  renders light energy similar to the one-photon absorption from ultraviolet (UV) light. NIR has been considered biologically safe unlike UV, which is harmful to soft tissues, cells and blood vessels. In addition to the heat and reactive oxygen species generating capability of 2PA molecules, 2PA-functionalized PCs were also found to be useful for treating diseases such as cancer by photothermal and photodynamic therapies. Herein, insights attained towards the design, synthesis and biomedical applications of 2PA-activated PCs are reviewed. In particular, specific focus is provided to the imaging and drug delivery applications with a special emphasis on multi-responsive platforms. The wide possibilities and realization witnessed in engineering a variety of structures and properties have made polymers a promising carrier matrix for functional biomedical applications such as drug delivery and imaging. The recent advances in modern polymerization techniques enables the synthesis of well-defined polymers with precise control over chemical composition, molecular weight, architecture and other functional properties such as stimuli-responsivity. Various strategies have advanced the biomedical applications of polymer-based drug delivery and imaging platforms, including chemical conjugation of active agents to the polymer scaffold, or physical encapsulation of active agents into self-assembled polymeric carriers (PCs) such as nanocapsules, micelles and polymersomes. Compared to free active agents, these PC-based systems endow several advantages, including improved solubility, long circulation and targeting ,2,3. MorLight as external medical stimulus is an easy and convenient tool useful for noninvasive therapy, image guided surgery, control over spatial resolution, localized polymerization and degradation of tissue engineering scaffolds ,11. When0) to a temporary virtual state of higher energy. Before relaxing back to its initial state, the system immediately absorbs the second photon and gets excited to the real final electronic state (S1). When an excited electron returns to the ground state, the generation of a photon with energy greater than either two of the absorbed photons occurs. This phenomenon is known as 2PA . I. I44]. IConjugated polymers (CPs) consist of alternating σ and π-bonds on their polymer backbones, and thus, electrons can delocalize throughout the entire polymer chain, which produces interesting and specific photophysical and electrical properties. Compared to the traditional fluorescence materials, CPs show several distinguished features such as large extinction coefficient, high fluorescence efficiency and excellent photostability. In general, CPs are highly hydrophobic, and thus, to realize these materials for in vivo biomedical applications requires significant improvement of their aqueous solubility. In this direction, the following two strategies have been considered . The firMcNeil and coworkers prepared CNPs from a series of CPs  derivative) by a reprecipitation method A 46]. T. T46]. TOwing to the tunable physicochemical characteristics and facile incorporation of photo-responsive functional groups, PCs have attracted much interest in the field of light-responsive targeted drug delivery systems. In general, most of the photo-responsive moieties incorporated into PCs respond to UV or visible light, which has limited penetration depth and thus cannot induce photoresponsive changes to trigger drug release. However, as an exogeneous stimulus, two-photon light irradiation can penetrate more deeply into biological tissues and facilitate the precise spatiotemporal control of drug release.−1. The IC50 was 147.8 μg mL−1 for chemotherapy and 36.2 μg mL−1 for PTT. The combination index (C.I.) is 0.48 (<1), which indicates the synergistic effect for chemotherapy and PTT. By taking advantage of the DNQ group transformation, Sun et al. also constructed a two-photon-sensitive and sugar-targeted nanocarriers from degradable and dendritic amphiphiles [Among the various photoresponsive functional groups, DNQ is one of the most extensively studied units. This functional group induces the solubility switch and triggers drug release from the PCs. Goodwin et al. synthesized a functional PEG-lipid nanoparticle with 2-diazo-1,2-napthoquinones (DNQ) . When thhiphiles .tert-butyldimethylsilyloxy)-coumarin (TBS-coumarin) derivatives in both solution and incorporated into PMMA matrix [The coumarinyl ester can be more easily cleaved by two-photon NIR light compared to the o-nitrobenzyl group. Hartner et al. studied the use of 2PA-induced cycloreversion of photodimerized 7-, these micelles triggered the release of DOX in cancer cells through photoisomerization of azobenzene via a two-photon excited fluorescence energy transfer process, resulting in effective antitumor therapy. Oleincizak et al. synthesized a two-photon degradable copolymer containing photocleavable moieties on the backbone by polymerizing adipoyl chloride, 1,6-hexanediol and a light-sensitive monomer with high two-photon absorbing efficiency [Using a popular light-sensitive azobenzene moiety, Huang et al. reported supramolecular conjugated unimicelles for two-photon-triggered drug release . These uficiency . The modo-nitrobenzyl groups on the side chain were prepared using poly(phenylene ethynylene)-based semiconducting polymer backbone, which also served as an upconversion agent [Apart from the delivery of small molecule drugs, multifunctional polymeric gene delivery carriers assisted with two-photon irradiation have been developed for the precise and effective treatment of both acquired and genetic diseases ,59. In ron agent . The denon agent .Instead of utilizing positively charged CPs, few studies have conjugated two-photon active agents to cationic polyethyleneimine (PEI), another widely used gene carrier, for efficient two-photon-assisted gene delivery. For example, Hayek et al. labeled PEI (25kDa) with a bis-stilbenyl-based two-photon active dye molecule and employed for gene delivery and imaging . The dyePDT is a minimally invasive treatment approved for certain cancers and non-cancerous disease states. It involves the administration of a photosensitizer, which is activated by light at specific wavelength, and generates cytotoxic species in the presence of oxygen.Luo et al. prepared a thermosensitive nanocomposite hydrogel from methoxy poly(ethylene glycol)-polylactide copolymer (mPEG-PDLLA) and Pluronic F127, which is approved for clinical use . A two-pβ-CD copolymer as the carrier, a zinc phthalocyanine as a photosensitizer and a tailored nitroaniline derivative as a nitric oxide photodonor [1O2 and NO and resulted in death of carcinoma cells.Kandoth et al. developed a three-component self-assembled polymeric nanosystem comprising an epichlorohydrin-otodonor . When enAs discussed in the previous sections, PCs equipped with the 2PA probe enable imaging and drug delivery capabilities and PDT. Recently, significant efforts have been devoted to integrating the above modalities into one single theranostic platform. These integrated systems may allow for a safe, efficient and minimally invasive tool for personalized medicine. In this section, we will discuss a few representative theranostic polymeric systems for two-photon bioimaging, chemotherapy, PTT and PDT.To date, polymeric nanoparticles are widely studied for cancer theranostics. Wang et al. prepared hybrid nanogels by immobilizing graphitic carbon dots (GCDs) within the PC matrix comprising a crosslinked PEG network and interpenetrated chitosan chains via an aqueous one-pot surfactant-free precipitation polymerization . The bio2) was linked to an aldehyde-end functionalized poly(methacryloyl phenylboronic acid pinacol ester-co-TPMA)-b-poly(2-azepane ethylmethacrylate) (CHO-P(MPA-co-TPMA)-b-PAEMA) to introduce an acid-labile benzoyl imide bond on the main backbone. Second, the drug Cap was conjugated to MPA unit via ROS-responsive phenylboronate ester bond to obtain the final amphiphilic prodrug copolymer (PMMTAb-Cap), which can form self-assembled micelles under aqueous conditions (b-poly(L-glutamic acid)-selenide micelles equipped with AIE-active two-photon probe were prepared and physically loaded with curcumin (Cur), a well-known herbal compound that exerts several anticancer effects in various types of cancers [Y. Wang and co-workers developed several polymeric systems that are capable of simultaneous two-photon imaging and stimuli-responsive drug delivery. By employing two-photon AIE-based imaging probe and judiciously placing stimuli-responsive units within the amphiphilic block copolymers either at the main backbone or side chain, they prepared number of theranostic polymeric platforms for enhanced two-photon imaging and effective therapy. Using the combination of RAFT polymerization and chemical conjugation methods, they synthesized a prodrug block copolymer featuring a two-photon AIE fluorophore and an anticancer drug capecitabine (Cap), which has been applied for the treatment of multiple malignant tumors, alone or in combination regimens . In brienditions . The TPM cancers . These m cancers . In vivoβ-CD via a ROS-responsive peroxalate ester to obtain a conjugate TPCD. Second, prednisolone (PRDL), a poorly soluble anti-inflammatory glucocorticoid, was encapsulated into β-CD via supramolecular interaction (b-PMPC (PMM) to form the theranostic micelles (TPCDP@PMM). The outer polymeric PMM shell can improve in vivo blood circulation and provide a chance for the accumulation of these micelles at the atherosclerotic lesion through the damaged vascular endothelium. The in vivo two-photon bioimaging of TPCD@PMMA on atherosclerotic ApoE-/- mice exhibited stronger fluorescence and more plaques were recognized. This confirmed the continuous accumulation of micelles. After accumulation, the overexpressed ROS level at the inflammatory tissue triggers a hydrophobic-to-hydrophilic switch of the PMEMA segment of the copolymer, leading to the disassembly of the structure. This process exposes the TPCDP to a high level of ROS and disrupts the peroxalate ester bond and facilitates the release of PRDL as well as the β-CD, resulting in two-pronged therapy via anti-inflammatory activity of PRDL and lipid removal feature of β-CD for atherosclerosis inhibition. These results suggest that these micelles can be promising nanoplatform for atherosclerosis theranostics. By employing a polymeric β-CD conjugate and a nitric oxide (NO) photodonor, a multifunctional biocompatible nanoconstruct has been synthesized for photo-controlled NO release with two-photon fluorescence imaging [β-CD conjugate using β-CD with epichlorohydrin under alkaline conditions, the NO photodonor with anthracene and nitroaniline structural unit was loaded into the polymeric conjugate to obtain the nanoconstruct. The NO release from the nanoconstruct was triggered by two-photon excitation using NIR laser (700 nm). The uncaged fluorescent co-product acted as a two-photon emission fluorescence reporter for the concomitant NO release. The release of NO in human squamous carcinoma cells was monitored by two-photon NIR fluorescence microscopy, which indicated the localization of fluorescent reporter in the cytoplasm. This nanoconstruct has potential for combined photo-activated therapies and bioimaging.Atherosclerosis is the gradual narrowing of the arteries caused by the buildup of plaque in the inner lining of an artery. It is one of the leading causes of death worldwide. The rupture of vulnerable atherosclerosis plaque is the major cause of sudden deaths. Therefore, imaging the structural and anatomic features of atherosclerotic plaque is crucial to inhibit the vulnerable atherosclerotic plaque progress. Recently, Ma et al. prepared supramolelcular theranostic micelles for combined two-photon AIE imaging of atherosclerosis diagnosis and two-pronged therapy . These meraction . Finally imaging . After so-nitrobenzyl were carefully placed within the polymeric nanoparticles to trigger on-demand photo-regulated drug release for enhanced therapy. Most of these systems are limited to cancer therapy. Thus, more two-photon-based systems should be designed and evaluated for other diseases. Despite the potential benefits and numerous advantages of these two-photon-activated polymeric systems, a number of studies conducted were mostly limited to in vivo animal models. The findings from animal studies are often difficult to transfer to humans. Therefore, studies related to preclinical efficacy and safety in humans are needed. Nonetheless, it was evident from the literature that 2PA responsive bioimaging can be effectively combined with other stimuli-responsive drug delivery and therapeutic applications. Additionally, it is certain that, in the future, the prime focus of 2PA-based research will move towards the development of a multifunctional theranostic system capable of performing imaging, drug delivery and ROS-induced therapy.The capability of excitation light utilized in 2PA on penetrating deep tissue without damaging normal tissue is providing impetus for the development of two-photon-based systems for biomedical applications. By integrating diverse modularly designed copolymers and two-photon AIE probe or conjugated polymers with large 2PA cross section, several polymeric nanoprobes have been developed for imaging various diseases. In most of the cases the well-resolved images were obtained with a tissue penetration depth of 150 µm. With appropriately designed two-photon probe with a large two-photon absorption cross section, under NIR-II excitation, small blood vessels as deep as 1064 μm in mouse brain are visualized. In recent years, 2PA-based bioimaging has been combined with nanoplatforms that perform smart drug delivery and ROS-mediated PDT. In most cases, NIR radiation of 800 nm was used as the 2PA excitation light. The combination of 2PA-based PDT and stimuli-responsive chemotherapeutic drugs have rendered effective cancer treatment. The photo-responsive functional moieties such as DNQ, coumarin and


The Oral versus Intravenous Antibiotics for Bone and Joint Infection Trial concluded that oral antibiotics administered during the first six weeks of therapy for orthopedic infections were non-inferior to parenteral antibiotics. Previously, we also conducted a retrospective cohort study of patients at our center with an orthopedic infection. Patients transitioned to oral therapy had significantly fewer adverse events and similar incidences of one year treatment failure compared to patients maintained on parenteral vancomycin.To increase use of oral antibiotics for orthopedic infections, we created a protocol for changing IV to PO antibiotics prior to hospital discharge. We evaluated its effect on prescribing practices and healthcare utilization outcomes. Patients with an orthopedic infection were included if they were discharged within the 90-day pre- and post-protocol study periods, had an inpatient Infectious Disease consult, and were initiated on parenteral vancomycin while inpatient. The primary outcome compared the incidence of oral antibiotics prescribed at discharge. Secondary outcomes included incidence of adverse drug reactions, discharge dispositions, hospital length of stay, canceled follow-up appointments, unplanned readmissions, and early treatment failure.Fifty patients were included, 22 and 28 patients in the pre- and post-protocol groups, respectively. Twenty-three (82%) patients in the post-protocol group were prescribed oral antibiotics at discharge compared to 8 (36%) patients in the pre-protocol group. Twenty-three (82%) patients in the post-protocol group were discharged home compared to 13 (59%) patients in the pre-protocol group. Of note, 7 (25%) patients in the post-protocol group had early treatment failure compared to 1 (5%) patient in the pre-protocol group, and 8 (29%) patients in the post-protocol group compared to 4 (18%) patients in the pre-protocol group had an unplanned readmission.Providing physicians with an oral antibiotic protocol for orthopedic infections significantly increased the number of oral antibiotic prescriptions at hospital discharge, but the higher rates of treatment failure observed after protocol implementation are concerning and warrant further investigation.Russell J. Benefield, PharmD, BCPS-AQ ID, Paratek Pharmaceuticals: Grant/Research Support.