For years the gene therapy community has been striving to engineer gene vehicles facilitating persistent, preferably life-long, expression of therapeutic transgenes. With the development of stem cell genetic therapies using human immunodeficiency virus 1 (HIV-1)-derived lentiviral vector systems  [1–4] and therapy of hemophilia B exploiting vectors based on adeno-associated virus (AAV)  [5], just to mention a few among many emerging treatments, it is fair to say that the perseverance of vector engineers has paid off. Much is still to be learned; whether expression of virally delivered transgenes can persist for decades in patients is not known, but current vector systems featuring optimized promoters, insulator elements, codon-optimized transgene cassettes, and the unique capacity of viruses to deliver genetic cargo with efficacy and specificity may support long-term gene expression, lending support to further clinical translation.

Who would have thought, after all these years, that delivery approaches allowing high, but only short-term, gene expression would be suddenly a key interest to the gene therapy community? As classical gene therapy approaches continue to develop, it will not have escaped anyone’s attention that genome editing and tools to engineer the genome are currently revolutionizing genetics and are quickly moving toward clinical use. Unique molecular tools that operate within the genome, like Cre and Flp recombinase, have been around for a while, but these are relevant as research tools only and not for clinical purposes due to the lack of compatible sequences in mammalian genomes. With the discovery of transposases capable of mobilizing genes with high efficacy in human cells, it became possible to integrate transgenes into genomes using nonviral delivery approaches  [6,7], essentially what was until then achieved by retroviral vectors carrying their own integrase proteins. Although the integration profiles of the most popular transposon vector systems based on the Sleeping Beauty and piggyBac transposons were found to differ markedly from the gene-biased integration profiles of gammaretroviral vectors and lentiviral vectors [8,9], this type of intervention lacks control and does not allow targeted insertion or engineering of the genome at predetermined positions. The game changed with the invention of zinc-finger nucleases (ZFNs) [10], consisting of a DNA-binding domain, designed to recognize a double-stranded DNA duplex with a certain sequence, fused to the endonuclease domain of the restriction enzyme FokI. Only by co-administration of two ZFNs recognizing two neighboring sequences a FokI dimer is formed upon binding of the ZFNs to the DNA, leading to creation of a double-stranded break (DSB) at this genomic position. It is now textbook knowledge that cellular DSB repair processes based on non-homologous end-joining (NHEJ) introduce indels at the cleavage site or that homology-directed repair of the damaged DNA using homologous DNA as a sequence donor may restore the sequence and potentially correct nearby mutations. This opened a world of opportunities, but it turned out to be challenging to fulfill the full potential of the ZFN technology due to difficulties related to engineering of ZFNs, leading to high costs and reduced accessibility and availability of effective ZFN pairs. Some of these challenges were circumvented with the invention of Transcription activator-like effector nucleases (TALENs) containing customizable array of repeats that can be assembled using easy-to-follow design guidelines [11,12]. However, with the discovery of CRISPR-directed adaptive immune responses in bacteria [13] and the implementation of RNA-guided endonucleases in human cells [14], targeted genome editing and introduction of knockout mutation with the speed of light has become a standard technique in molecular genetics research. Hence, cellular delivery of the endonuclease (typically Cas9 from Streptococcus pyogenes) guided by a single guide RNA (sgRNA) molecule facilitates firm studies of causal linkages between genetic variation and cellular phenotypes, but numerous other applications of the CRISPR system, far too many to discuss here, appears with constant pace. Although far from trivial, one of the ultimate goals is to deliver components of the CRISPR system to individuals aiming at treating disease by genomic editing directly in the patient.

From old-school, but still highly relevant recombinases and transposases, to ZFNs and a wide variety of advanced and user-friendly RNA-guided DNA and RNA endonucleases, the genomic tool box is rapidly expanding in the hands of genome entrepreneurs. Common for the use of all these tools is that efficient delivery across cell and nuclear membranes is essential for efficacy. As new tools appear, they are traditionally tested in proof-of-principle studies showing efficacy after delivery of plasmid DNA or viral vectors encoding the particular protein. Using methods based on intracellular production of the relevant tool, the genomic engineering community has directly benefitted from years of accumulated experience with gene therapy vectors aiming at establishing persistent and highest-possible levels of gene expression. However, long-term expression of recombinases or DNA endonucleases may potentially be harmful to the genome.
Intuitively, an optimal vehicle for delivering genome editing tools results in a short-lived boost of enzyme activity, long enough to do the job and short enough not to cause unwanted genomic modifications or off-target effects. At the same time, the short boost should not compromise specificity and safety due to momentary far too high protein and activity levels. These goals have an inherent contradiction in terms, since high-efficacy delivery is likely to cause unnecessary high levels of protein that may last longer than required for the desired genomic modification to occur. Therefore, editors of the genome are facing a new set of challenges calling upon new ways of delivering nucleic acids and proteins to cells or tissues. Transfection of in vitro-transcribed mRNA encoding transposases or endonucleases is a well-established approach, which results in short-term intracellular expression due to the short half-life of mRNA. This method has been successfully used for delivery of transposases, ZFNs, and TALENs to primary T cells [15–17] and hematopoietic stem cells [18] and is clinically applicable [19]. Despite evidence of in vivo applicability based on transfection of mouse liver by hydrodynamic injection [20,21], administration of mRNA seems to have limited potential for in vivo use. The same may be true for delivery of recombinant protein, although delivery of in vitro-synthesized spCas9 protein in ribonucleoprotein (RNP) complexes consisting of recombinant spCas9 and in vitro-synthesized sgRNA is now well established in cultured cells. Delivery of recombinant protein can be assisted also by conjugation with cell-penetrating peptides (CPPs) [22,23], but yet other proteins, like the Sleeping Beauty and piggyBac transposase do not seem to be compatible with in vitro production methods, and earlier attempts to deliver such proteins to cells have been unsuccessful [24,25]. In the past few years, a new protein delivery approach based on the incorporation and transport of heterologous proteins in the HIV-based lentiviral particles has emerged. As it seems, lentivirus particles are sufficiently flexible to allow packaging and processing of nonviral proteins, and proteins ranging from transposases and designer nucleases to RNA-guided endonucleases can be delivered to cells as cargo transported by lentiviral couriers. In this Expert Insight article, I review the repurposing of lentiviral vectors for delivery of proteins for genome engineering.

Lentiviral delivery of vector-encoded genome editing tools

Lentiviral vectors based on HIV-1 were developed for delivery of gene expression cassettes using strategies that were basically adapted from gene vectors based on genetically simpler viruses like the gamma-retroviruses with murine leukemia virus (MLV) as one of the primary drivers in the field. Production of the first generation of lentiviral vectors was based on co-transfection of the transfer vector plasmid with plasmids encoding the vesicular stomatitis virus glycoprotein (VSV-G) and the viral proteins including all accessory proteins like Rev and Tat. The resulting vectors were found to transduce nondividing cells [26] and, thus, offered an attractive alternative to MLV-based vectors. Due to the risk of generating replication-competent HIV-1, both the transfer vector and packaging plasmids later underwent considerable modifications. Hence, for production of third-generation vectors, now widely used in laboratories worldwide, expression of full-length vector RNA from the transfer vector plasmid is driven by a CMV promoter rather than by the natural HIV-1 promoter. This means that transcription does not require the Tat accessory protein. Also, natural HIV-1 promoter sequences have been deleted from the U3 region of the vector, ensuring that the viral promoter is not present in the reverse-transcribed and eventually genomically inserted vector sequence [27,28]. This type of vector is referred to as a self-inactivating (SIN) vector. Equally important, the safety of the system was further improved by removal of sequences encoding the accessory proteins from the packaging construct. The gene encoding the Rev protein, which is required for proper nuclear export of the vector RNA, was moved to a separate plasmid, resulting in a production scheme based on co-transfection of four plasmids, (i) the SIN vector plasmid encoding the transgene of interest driven by an internal promoter, (ii) the GagPol-encoding packaging construct, (iii) a Rev expression plasmid, and (iv) a plasmid encoding VSV-G. Limited sequence similarity between these four plasmids and the absence of a subset of viral genes rendered production of replication-competent HIV-1 very unlikely [29]. Even with such modifications requiring co-transfection of several plasmids, production schemes are efficient, and co-transfection of an easy transfectable cell line, like HEK293T, results in standard production of millions and millions of virus particles each loaded with a vector RNA dimer. Due to the broad tropism and high gene transfer efficacy, VSV-G-pseudotyped lentiviral vectors have become a state-of-the-art tool for delivery of gene expression cassettes to cell lines and hard-to-transfect primary cells. It is beyond the scope of this article to cover the many aspects and applications of lentiviral gene delivery, but genetic therapies based on this technology are obviously attracting particular attention [30,31]. Emerging evidence supports clinical use of lentiviral vectors for correction of hematopoietic stem cells for treatment of primary immunodeficiencies, leukodystrophies, and hemoglobinopathies [3,32–34], and corrective ex vivo gene therapy was recently approved in Europe for treatment for ADA-SCID [35,36]. With big pharma moving into the gene therapy area, one example being GlaxoSmithKline acquiring the commercialization rights to Strimvelis for ADA-SCID treatment, additional treatments are likely to gain approval in the near future. Notably, this first licensed gene therapy using an integrating vector system is based on a gamma-retroviral vector system. A comprehensive overview of current hematopoietic stem cell gene therapies in clinical development can be found in a recent review [37].

The capacity to transfer and genomically integrate genetic information makes lentiviral vectors powerful carriers of genome editing tools. However, in contrast to the goals of conventional gene therapies, prolonged expression of endonucleases is rarely desired for therapeutic genome editing purposes. Gene transfer by integrase-defective lentiviral vectors (IDLVs; reviewed in  [38]), vectors that carry an inactive integrase protein, does not lead to gene insertion, and, thus, allows only transient transgene expression in dividing cells. We have previously shown delivery of recombinases and transposases using an IDLV-based platform [39–41]. Also, IDLV-based delivery of ZFN expression cassettes has been demonstrated on several occasions [42–45], whereas a similar strategy for delivery of TALENs is not applicable due to frequent recombination between repeated sequences in the TALEN gene [46]. However, this problem can be overcome by altering codon sequences [47] or by expressing the TALEN protein from RNA delivered in lentiviral particles that do not support reverse transcription due to lack of a functional reverse transcriptase enzyme [48]. Other potential RNA delivery methods exploit an MS2-driven packaging system, allowing lentivirus-based transfer of heterologous RNAs [49]. For delivery of Cas9 and sgRNA cassettes, transfer by lentiviral vectors is now well established [50,51] and can be harnessed for gene knockout or repair essentially as shown schematically in Figure 1. Exploiting the capacity to integrate the Cas9 gene as well as the sgRNA gene, such vectors are widely used for functional genomics and genetic screens based on genome-wide CRISPR-directed gene knockout [50–54]. However, data supporting effective IDLV-directed delivery of the CRISPR/Cas system is still lacking in the literature. Notably, episomal DNA intermediates formed in IDLV-transduced cells serve as effective templates for repair by homologous recombination, supporting the use of IDLVs as vehicles of genome editing repair substrates [42–44,55,56].

Re-thinking lentiviral delivery – now ferrying proteins

Try google ‘repurposing’ and somewhere on the Internet you will find that ‘repurposing is the art of finding new uses for old items’. That works for an old wheelbarrow carrying your garden flowers or a chair made of your old alpine skis. But does it work for lentiviral vectors? A few years back, we initiated work aiming at repurposing lentiviral particles for delivery of genome-modifying proteins like transposases and site-targeted endonucleases. Our ultimate goal was to explore the capacity of viruses to carry and transfer both scissors for genomic surgery and the donor patch for repair by homology-directed repair (Figure 2). Early work aimed at tracking intracellular migration of the HIV core exploited fusion of a reporter protein, like GFP, to the Vpr accessory protein  [57]. As previously reviewed in [58], a similar strategy was used for packaging of different types of proteins in lentiviral particles, but toxicity of Vpr in transduced cells is a potential problem [59]. Inspired by work of Jun Komano and coworkers, we decided to focus on protein incorporation strategies based on fusing proteins of interest to the lentiviral Gag and GagPol polypeptides. Initially, Aoki et al. fused β-lactamase to the N-terminal end of the Gag polypeptide and added the myristoylation signal of lyn to the N-terminal end of β-lactamase [60]. Enzymatic activity was detected in cells exposed to the resulting lentivirus particles, each loaded with about 5000 copies of β-lactamase. A similar approach was used subsequently to induce apoptosis in cells treated with lentiviral particles carrying caspase 3 protein fused to the matrix protein in the N-terminal end of Gag and GagPol [61].

Incorporation of genome-modifying proteins into lentiviral particles by fusion to the Gag and GagPol polypeptides is based on the accumulation of Gag and GagPol at the inner surface of the cell membrane during virus assembly. As depicted schematically in Figure 3, Gag and GagPol polypeptides, tagged N-terminally with a protein of interest, are expressed from a packaging construct transfected into virus-producing cells. Alongside this plasmid, the cells are transfected also with plasmids encoding the virus envelope protein (typically VSV glycoprotein) and the Rev protein (not shown). Also, plasmid DNA encoding packagable vector RNA carrying either a DNA transposon sequence or a donor sequence for homology-directed repair can be included in the transfection mix. Budding lentivirus particles consist of approximately 5000 Gag molecules and about 250 GagPol molecules [62], the latter generated only through a frameshift at the Gag-Pol junction. If vector RNA is expressed in the cell, a vector RNA dimer will be encapsidated in the particle through interactions between the nucleocapsid [NC) protein and the packaging signal located near the 5’ end of the RNA. Released virus particles undergo a maturation process, which is based on the cleavage of Gag and Pol domains into structural and enzymatic proteins, respectively. This process is catalyzed by the viral protease, which releases the foreign fusion protein as the core condenses in the maturating particle. Proper protein release is achieved by introducing a HIV-1 protease recognition site at the junction between the tagged protein and Gag. Upon uptake by endocytosis in transduced cells and subsequent endosomal escape, released proteins eventually enter the nucleus through unknown mechanisms that may involve migration as part of the virus core or diffusion through the cytoplasm.

Gene insertion by lentiviral particles loaded with transposases

With resurrection of the Sleeping Beauty DNA transposon back in the late 1990s [6] and evidence of effective piggyBac DNA transposition in mammalian cells  [63], it became possible to insert transgenes into mammalian genomes using nonviral delivery platforms, typically plasmids encoding a transposon-embedded transgene cassette and the transposase. Others and we showed preclinical efficacy of gene delivery by DNA transposition in animal models [7,64–66], and efforts were later made to deliver the two-component vector system using viral vehicles  [39,41,67]. Also, DNA transposition has been achieved by administration of in vitro-transcribed mRNA [15,20,21], but attempts to purify and administer recombinant Sleeping Beauty and piggyBac transposase protein have not been successful [24,25]. In proof-of-principle studies of lentiviral incorporation and delivery of protein, we therefore initially focused on loading of DNA transposases into lentivirus particles. Utilizing a third-generation packaging construct, as depicted in Figure 3, we produced virus particles carrying a hyperactive piggyBac transposase (hyPBase, originally described by Yusa et al. [68]) fused via a protease cleavage site to the N-terminus of Gag and flanked on the N-terminal side of the Lyn-derived myristoylation signal. Upon virus particle maturation, this stowaway protein was released from the polypeptide and induced high levels of DNA transposition in cells treated with the virus [69]. Notably, we also learned that virus particles carrying Gag/GagPol with an N-terminal fusion domain were not able to deliver and reverse-transcribe vector RNA. To circumvent this restriction, we generated mosaic viruses carrying wildtype Gag/GagPol as well as hyPBase-fused Gag/GagPol in each particle and demonstrated the capacity of such viruses to co-deliver vector RNA. In fact, by engineering vector RNA carrying the transposon sequence, which upon reverse transcription would become a substrate for DNA transposition, we observed effective mobilization of the transgene in cells treated with ‘all-in-one’ IDLVs carrying both hyPBase and the transposon. Such DNA transposition was evident in different cell types including a panel well-known cell lines and primary cells, like keratinocytes and dermal fibroblasts. By staining of virus-treated cells for the hyPBase (using an HA-tagged version of the transposase), we found only low levels of the transposase protein as opposed to the high levels detected in cells transfected with hyPBase-encoding plasmid DNA. Still, levels of DNA transposition, as measured by colony formation after mobilization of a puromycin resistance gene, were equally high. Although still hypothetical, this could indicate that protein delivered by the virus does not rely on random diffusion but rather traverses the cytoplasm and the nuclear membrane through mechanisms that are supported by the intracellular movement of the viral core. If such mechanisms are indeed in play, this would argue that virus-delivered enzymes can be effective even at relative low intracellular concentrations.

Genome editing by lentiviral delivery of ZFNs or Cas9 protein

Prior to the arrival of CRISPR on the genome editing scene, we investigated the potential of lentiviruses as carriers of designer nucleases [70]. Using a slightly modified strategy exploiting a packaging construct carrying the heterologous phospholipase C-δ1 pleckstrin homology (PH) domain for improved Gag/GagPol recruitment to the membrane, pairs of ZFNs were incorporated into lentivirus particles. ZFN-loaded virus particles were produced by cells transfected with two packaging constructs, each carrying a ZFN sequence fused to the gag gene via the PH domain. Incorporation and protease-dependent release of was verified for both ZFNs, and DNA cleavage leading to indel formation was obtained in cell lines and primary cells after delivery of ZFN pairs targeting a transgenic egfp locus, the CCR5 locus, or the AAVS1 locus. In case of the most effective set of ZFNs (targeting CCR5), targeted disruptions were detected in one fourth of the CCR5 alleles in a population of primary keratinocytes treated with ZFN-loaded lentiviral particles [70]. By co-packaging of vector RNA carrying a template for homologous recombination in ‘all-in-one’ IDLVs stuffed with egfp-directed ZFNs, repair of a mutated egfp report gene was achieved in more than 8% of the cells in a HEK293 cell-based model, indicating that protein and vector RNA could be effectively co-delivered. This approach was later utilized to achieve site-directed transgene insertion into the CCR5 and AAVS1-loci in hematopoietic progenitor cells and induced pluripotent stem cells (iPSCs) [71]. Notably, 34 out of 38 analyzed iPSC clones generated by treatment with ZFN-loaded lentiviral particles carried the reporter gene cassette inserted precisely by homologous recombination into the locus that was targeted by the ZFNs.

The optimal tool for genome work is immediately effective and short-lived. By administering the protein itself and not the source for production of protein, the genome-modifying activity will only last until the protein is diluted and degraded. Immunostaining of cells treated by lentiviral ZFN protein transduction showed detection of ZFNs already within the first hour after exposure to the virus and certified that the protein was gone (or at least not detectable) after twenty four hours [71]. Intuitively, such time restriction should improve the safety of the procedure and be beneficial as long as the activity is high immediately after delivery. In fact, comparing a transfection-based ZFN delivery approach and virus-directed ZFN delivery for targeted disruption of the CCR5 locus, we found reduced off-target cleavage within the neighboring CCR2 locus after protein delivery even under experimental conditions facilitating higher on-target rates than observed by plasmid transfection  [70]. In support of this, next-generation sequencing of a number of potential off-target sites in cells harboring a targeted transgene insertion obtained through viral ZFN delivery did not show any sign of off-target cleavage [71].

Successful delivery of active protein by engineered lentiviral particles depends on proper cleavage of the Gag/GagPol polypeptides, leading to release of the protein during virus particle maturation. In case of both transposases and ZFNs, cleavage happens to occur specifically at the protease cleavage site between the fusion domain and Gag, but we have also seen examples of proteins that are cleaved internally by the viral protease, despite the apparent lack of a HIV-1 protease cleavage site at this particular position. Such internal cleavage was evident for egfp-directed TALENs, resulting in only few particles carrying the full-length protein and reduced targeted disruption rates in virus-treated cells [70].

The probability of proteolytic cleavage at positions inside the protein of interest supposedly increases with the size of protein, but we suspect that also the protein structure can be decisive for protease accessibility and potential cleavage. With the protein size in mind, we initially invested much effort in incorporating the Cas9 nuclease from Staphylococcus aureus (saCas9; [72]) into lentiviral particles using the above-described approaches. SaCas9 is 1053 amino acids long and, thus, 315 amino acids shorter than Streptococcus pyogenes Cas9 (spCas9). However, we only detected limited amounts of full-length saCas9 in mature lentiviral particles, resulting in low levels of targeted gene disruption in virus-treated cells expressing an appropriate sgRNA (Y Cai and JG Mikkelsen, unpublished observations). Surprisingly, however, spCas9 fused to the N-terminal end of Gag could be effectively packaged in lentiviral particles and was released from Gag primarily as a full-length 160 kDa protein during maturation of the particle [73]. It was estimated that 3 million particles contained a total of approximately 50 ng spCas9 protein. By transduction of primary T-cells first with a lentiviral vector encoding a CCR5-directed sgRNA and then with spCas9-loaded viral particles, indels in CCR5 were generated in 15% of the cells. Furthermore, co-delivery of spCas9 protein and a vector encoding a sgRNA directed against the CD4 gene in an ‘all-in-one’ format resulted in targeted CD4 disruption and reduced presentation of CD4 on the surface of HeLa-derived TZM-BL cells  [73]. Also, by including a sgRNA targeting HIV long terminal repeats (LTRs), spCas9-loaded virus particles induced excision of the HIV proviral DNA in a T-cell line harboring a transcriptionally competent HIV-1 provirus  [73].

Translational insight

Over the last 20 years, lentiviral vector systems have been developed and optimized for gene therapy applications. More than ever, it seems that therapeutic gene transfer using HIV-1-derived particles will benefit patients and therefore continue to attract attention of investors. For genome editing purposes, persistent expression of essential tools, like Cas9 and sgRNA, is attractive only for certain applications, like genome-wide CRISPR screens, and short-lived activity of site-targeted endonucleases is desired, at least for potential clinical use. For ex vivo applications, including editing in genomes of cultured stem cells, delivery of RNPs consisting of recombinant Cas9 protein and synthetic sgRNA stands out as a robust and relatively safe administration strategy [74,75]. For in vivo genome editing, however, it is not currently obvious how genomic tools are delivered effectively without compromising safety, although recent examples of delivering Cas9/sgRNA gene cassettes by use of adeno-associated virus-derived vectors show solid targeted gene disruption in mouse liver [72].

In attempts to repurpose lentiviral vectors as a delivery tool, others and we have studied the capacity of lentiviruses to encapsidate and transfer foreign protein of nonviral origin. In fact, the structure of the lentiviral particle seems more flexible than one should have thought considering the evolutionarily conserved structure and function of the enveloped capsid. In any case, the list of proteins successfully incorporated in lentiviruses is growing (see reference [76] for recent review), and different protein packaging strategies have turned out to be effective. Hence, proteins can be fused to the N-terminal end of Gag (as described above) or to the integrase protein in the C-terminal end of GagPol [77–79], or inserted between the matrix and capsid proteins in Gag [80]. Regardless of the configuration, solid and time-restricted protein activity is observed in cells exposed to protein-loaded virus particles. As a unique feature protein-loaded particles can carry also a template sequence for homologous recombination, allowing co-delivery of complete genome editing tool kits in cells that take up the virus.

Furthermore, by incorporating heterologous envelope proteins in the envelope surrounding the viral core particle, it is possible to pseudotype lentiviral vectors and alter the tropism [81]. Just like delivery of genes can be targeted to specific cell types, pseudotyping can be exploited also to target genome editing to specific tissues or subsets of cells. A recent report demonstrating delivery of ovalbumin into antigen-presenting cells using lentiviruses pseudotyped with measles virus glycoproteins (using Signaling lymphocyte activation molecule, SLAM, as the primary receptor) serves as proof-of-principle for cell-targeted protein delivery using protein-loaded lentiviral particles [80]. Such targeted delivery of genome-editing tool kits utilizing the capacity of virus particles to circulate and target cells may pave the way for in vivo engineering of the genome through processes that are both cell- and locus-specific. Hopes are that efficacy and safety can go hand-in-hand toward preclinical and eventually clinical translation of repurposed lentiviruses – now transferring protein.

Acknowledgments

Research focusing on genome engineering technologies in the laboratory of JGM is made possible through support of the Danish Council for Independent Research & Medical Sciences (grant DFF-4004–00220), The Lundbeck Foundation (grant R126–2012–12456), the Hørslev Foundation, Aase og Ejnar Danielsens Fond, Grosserer L. F. Foghts Fond, Agnes og Poul Friis Fond, Oda og Hans Svenningsens Fond, Snedkermester Sophus Jacobsen & Hustru Astrid Jacobsens Fond, Familien Hede Nielsens Fond. JGM is head of Gene Therapy Initiative Aarhus (GTI-Aarhus) funded by the Lundbeck Foundation and a member of the Aarhus Research Center for Innate Immunology (ARCII) established through funding by the AU-Ideas program at Aarhus University.

Financial & competing interests disclosure

The author has no relevant financial involvement with an organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock options or ownership, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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Affiliations

Jacob Giehm Mikkelsen

Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark

This work is licensed under a Creative Commons Attribution- NonCommercial – NoDerivatives 4.0 International License.