5.2 A RTICLE II
5.2.4 Detection of viral particles by MRI
signal was found on the injected side of the rat brai detectable for at least two weeks (14 days) (II / strong tropism towards cuboid epithelial cells in the the accumulation of iron to these cells m
area of the rat brain. Even though the secretive endocytotic traffic (Emerich et
endocytosis of the bUSPIO-particles, since no receiving only bUSPIO suspension.
The administrated iron (500 ng) maximal concentration of 1,7 ng/µl
was small, as there have been studies with intracerebral administration of 25µg of iron (Muldoon et al., 2005). As compared to the reported detection limits of 10 µM, the concentration factor would be 1000-fold. Wh
fl (D
Baavi particles are likely to have esca
The dimishment of the MRI signal loss during time might be explained by CP epithelial cell turnover (Netsky and Shuangshoti, 1970; Chauhan and Lewis, 1979) or even cell proliferation as response to injury from the injection pressure (Li et al., 2002). After intracerebral administration the USPIO particles have been observed to accumulate in cervical lymph nodes via CSF efflux (Kida et al., 1993; Muldoon et al., 2004) which agreed with previous findings of baculovirus systemic escape to ectopic tissues (Lehtolainen et al., 2002) and data from later SPECT experiments (III / fig 3). These observations could explain the fate of free bUSPIO and bUSPIO-Baavi complexes.
The nonlinearity of the iron signal hinders the evaluati
Iron detection by Prussian blue staining
The relationship of the iron originated signal loss to the biodistribution ed by Prussian blue staining for iron. As the amount inobenzidine (DAB) signal enhancement for the detection (Moos a
thod resulted in staining of the cuboid epithelial cells of CP on the side of the injection, while the contralateral CP cells remained without any stain, similar to controls without any injected iron.
As the CSF flow originates from the lateral ventricles and passes ourth ventricle to the to superior sagittal sinus via the subarachnoid spac
liquid dynamics (Figure 13) are likely to prevent the diffusion of bUSPIO-virus pa ed ipsilateral ventricle to the contralateral ventricle. This would explain the lim of the coated virus.
5.2.5
of the baculovirus was
confirm of iron was low, we used
diam nd Mollgard, 1993). This
me
through the third and
f e (Knopf et al., 1995), the
rticles from the
inject ited diffusion
Figure 13. The flow of cerebrospinal fluid (CSF) in human brain, (a) coronal plane and (b) midsagittal plane.
The CSF is produced in ependymal cells of choroid plexus cells in lateral ventricles (1.) and it flows through the third ventricle (2.) to the fourth ventricle where it can continue to the spinal subarachnoidal space or (3.) continue through the lateral aperture of the fourth ventricle to the subarachnoidal space (4.) of the brain. The CSF in the subarachnoidal space moves to the superior sagittal sinus (5.) where it is reabsorbed via the arachnoidal villi into the venous system.
5.2.6 Viral transgene expression
In order to compare and examine the viral transgene expression, we performed β-galactosidase staining for the viral LacZ-transgene expression. As previously (Lehtolainen et al., 2002; Laitinen et al., 2005a), the β-galactosidase staining resulted in staining of cuboid epithelial cells of CP. Only a few blue cells were seen in the contralateral side, in agreement with the Prussian blue staining for iron. As compared to wild-type virus or uncoated Baavi, the results in ipsilateral side were similar or transduction efficiency was slightly increased, determined by the amount of blue cells. Since the USPIO coating increases the size of the virus, it is possible that larger sized complexes are more
easily endocytosed into the cells (Muro et al., 2004), as also suggested in a study which compared the uptake of different sized iron oxide particles (Raynal et al., 2004).
trast to the wild-type virus, some blue cells were detected sporadically in the
ination of the bUSPIO particles from CP, it would be interesting to determine if the similarity is caused by cellular regeneration.
inly in young children with 0.3 cases per a 1 million population. While surgical treatment is the primary option in the majority of the cases, the poor prognosis even with radio- and chemotherapy suggests that gene therapy could be used along with the traditional methods (Gupta, 2003; Wolff et al., 2002). There is also evidence that weight regulation via leptin interaction could be affected by numerous leptin receptors in CP (Strazielle and Ghersi-Egea, 2000), creating commercial potential for weight regulation by gene therapy.
In this light, the further research of the baculovirus mediated transduction of CP seems reasonable. The described MRI-based imaging has several benefits as compared to the traditional
In con
brain parenchyma with Baavi and USPIO-coated Baavi. It may be possible that the positive charge of the avidin on Baavi might enhance attachment and result in improved transduction of known permissible cell types, such as endothelial cells of microvessels (Lehtolainen et al., 2002), glial cells or astrocytes (Sarkis et al., 2000).
It is known that the expression of baculovirus transgene is reduced from 80 % at day 5 to almost zero at day 14 (Lehtolainen et al., 2002). Interestingly, the iron-related signal loss was also reduced to background in two weeks. Although short-time expression of baculovirus may be entirely separate from the elim
5.2.7 Choroid plexus as targets for gene therapy
This study showed that detection of viral particle biodistribution by MRI is possible. The data confirmed the co-localization of bUSPIO with Baavi and moreover indicated that Baavi could deliver cargo to CP cells. Magnetic nanoparticles have been utilized as drug carriers (Alexiou et al., 2006), with therapeutic results and decreased side effects (Alexiou et al., 2003). The combination of gene delivery and drug delivery together might be beneficial when considering the versatile role of CP in physiology.
CP are best known best for their role in producing CSF, but they also play a role in the immune system, maintaining the blood-brain barrier (BBB), detoxification, secretion of various molecules and neurogenesis (Emerich et al., 2005). Human CP cells produce 500 ml of CSF per day and are reported to be involved in various medical conditions, such as Alzheimer’s disease (Emerich et al., 2005), and in brain regeneration, containing neuronal precursor cells (Li et al., 2002). Therefore the possibility of affecting the central nervous system via the CSF by gene therapy would be intriguing. Interestingly, it has been suggested that 30% of CSF is produced at other sites than CP, such as the epithelial lining of the ventricles and the endothelium of the brain capillaries (Davson, 1972; Hammock and Milhorat, 1973). Together with the CP cells, all those sites have been found to be transduced with baculovirus (Lehtolainen et al., 2002; Kaikkonen et al., 2006).
Although this behaviour might be due to common extracellular properties deriving from embryonal properties (Sarnat, 1998), further research on this matter might also shed some light on to the baculoviral tropism and cell entry in the central nervous system (CNS).
According to literature, viruses such as Sendai-, mumps- and human T-cell leukaemia virus-1 and possibly HIV-1 have tropism for CP (Levine, 1987; Strazielle and Ghersi-Egea, 2000).
This might be explained by the role of CP in the neuroimmune system (Engelhardt et al., 2001), relaying information between the brain and the immune system (Lacroix et al., 1998b). Recently, CP transplants have been studied for neuroprotective potential and spinal neuron regeneration (Lacroix et al., 1998a). Ex vivo transduction of neuroprotective genes, such as VEGFs (Zachary, 2005) could be combined with such transplants to further enhance the neuroprotection.
CP tumours have a rare prevalence of 0.5 % of all brain tumours occurring ma
histochemical mechanisms. Especially when including a MRI-visible transgene system with ifferent relaxivity (Table 5) to the virus, both particle biodistribution and transgene imaging could
erfusion and metabolic and ctions (Yang and Atalar, 200 , this MRI-based baculovirus imaging system to further characterize novel t or CP related conditions.
d
be performed with the same device, although iron effects to detection of other contrast agents as well. Additionally, MRI is also capable of imaging physiological changes in organ p
mechanical fun
could be used 6). In time
reatments f
5.3 Article III
As discussed in the previous chapter, the transduction of choroid plexus in brain has so far showed promising results and set goals for further studies. As intracerebroventricular (ICV) administration is mostly suited to the treatment of brain disorders, there is still little knowledge of the baculoviral systemic kinetics in the body after ICV injection and traffic routes after other administration routes.
Since the baculovirus is hindered by the blood complement (Hofmann and Strauss, 1998), biodistribution studies based on the transduction pattern of the virus would not provide accurate
formation about the viral kinetics after administration, but tracking viral particles could provide dditional information about baculovirus systemic kinetics.
with biotinylated USPIO could create ution, the poor temporal resolution favour the
idney and a minor
99m
resulted b
virus with the labelled chelate did not show any activity. Despite the SPECT revealed activity in the spleen, no beta-galactosidase expression was detected with Baavi or wild-type virus.
in a
While the MRI- imaging of Baavi coated accurate information about brain particle biodistrib
use of other imaging method when acquiring data of rapid systemic changes in baculovirus administration kinetics. As discussed in 2.4.1.2, SPECT imaging has these advantages over MRI imaging, also enabling quantitation of the signal.
In this study, we compared different routes of administration of avidin-displaying baculovirus, Baavi, coated with polylysine-serine-DTPA chelate (III/ fig 1) with 99mTechnetium. By using a combined microSPECT/CT device the particle biodistribution could be monitored real-time in the SPECT with the ability to include anatomical references by CT imaging.
5.3.1 Intravenous administration
Systemic injection is the preferred method to use, when acquiring a wide access to organs by gene delivery vectors. The complement inhibition hinders the systemic use of baculovirus; however the inhibition is not always complete. Previously it has been shown that systemic administration of non-modified baculovirus through a tail vein injection in BALB/c mice resulted in GFP expression in liver, spleen, lung, heart, kidney and brains (Kim et al., 2006). Similarly, after systemic injection of A/J mice a significant lusiferase expression was seen in the spleen, liver and k
expression in the lungs (Kircheis et al., 2001). However, no GFP or luciferase expression was seen after intravenous administration of VSV-G displaying baculovirus into BALB/c mice (Tani et al., 2003b). While the results may be due to pseudotyping of virus, a variation in dose, rodent strain, individual properties of experimental animals or virus preparation between laboratories, there might also be other factors influencing to the expression pattern.
The SPECT planar imaging of Baavi coated with technetium-labelled chelate showed increasing activity after systemic injection via v. femoralis, (IF), (III/ fig 2) in the lungs, liver, spleen and kidneys while the imaging of the radiolabelled chelate or Tc alone (data not shown) in rightening of kidneys, ureters and bladder as the urinary export of water-soluble substances progressed. It has been shown that the reticulo-endothelial system of the liver plays an important role in the active disposal of administered viral particles (Tao et al., 2001), together with the spleen and kidneys, being therefore expected to gather virions. The signal seen in the lungs was decreased in time, likely representing viruses circulating in blood (Schellingerhout and Bogdanov, Jr., 2002).
Baavi resulted in moderate beta-galactosidase expression in kidneys and liver (III, table 1), while IF injections of the wild-type
5.3.2 traperitoneal and intramuscular administration
traperitoneal administration (IP) has been used for the treatment of ovarian cancer, in an attempt proved access to the peritoneal cavity has a direct access to the thoracic and
transduce various mammalian kidney cell lines in vitro (Liang et al., 2004;
Condreay et al., 1999). The reports of kidney transduction after systemic tail-vein administration do
l, although not clinically preferred method. Previously it has been reported that the intracranial administration of baculoviral vector has resulted in GFP expression in mouse and rat brain striatum, corpus callosum and ependymal layer neuronal cells
GFP expression in the mouse brain (Tani et al., 2003b), In
In
to overcome the limitations of intratumoral injections for im (Evans and Keith, 2004). However, the peritoneal cavity
pleural cavities via the lymphatic channels (bu-Hijleh et al., 1995a). The peritoneal fluid enters from the peritoneum to the lymphatic lacunae and continues onwards via the parasternal lymphatics to the superior mediastinal nodes (bu-Hijleh et al., 1995b; bu-Hijleh et al., 1995a). This access enables both ovarian tumour metastases and injected virus spread systemically through the lymphatic system, the latter being important for viral safety.
IP injection of the labelled Baavi resulted in significantly increased radioactivity in spleen and kidney (III/ table 1). Beta-galactosidase stainings of the spleen resulted in moderate expression of the LacZ transgene and in the kidney resulted in strong and extensive beta-galactosidase expression (III/ Figure 4). According to a study, baculoviruses have previously been shown to efficiently
not show extensive transduction in one study (Kim et al., 2006), while the other shows kidneys quite positive (Kircheis et al., 2001). It might be possible that avidin display has enhanced the baculovirus transduction mechanism as compared to the wild-type virus, as suggested by our previous study (I), to result in the successful transduction of the kidney after both IP and IF administrations.
After IP administration some beta-galactosidase expression could also be seen in the lungs and the brain and radioactivity was occasionally detected in mediastinal nodes during the SPECT imaging (data not shown). Interestingly, the brain was also seen unexpectedly positive after systemic administration in a previous study, increasing with the PEGylation of the baculovirus (Kim et al., 2006). This indicates that under suitable conditions the baculovirus is possibly able to transport across the BBB and transduce brain parenchymal cells with better capability than previously thought. Our results suggest that after IP injection the baculovirus enters the lymphatic circulation from the peritoneal lymphatic drainage. Similarly after intramuscular injection with Baavi, we could detect the femoral lymphatic nodes being imaged, indicating viral traffic via the lymphatic system. Some transgene expression could also be seen in the kidney cells (data not shown), indicating that Baavi is also likely to escape to the systemic circulation after intramuscular administration. Previously it has been shown that intramuscular administration of baculovirus results in successful transduction of mouse skeletal muscle (Pieroni et al., 2001).
5.3.3 Intracerebroventricular administration
In order to bypass the BBB and to provide access for drug or gene delivery vector administration by ICV injection has provided a usefu
(Sarkis et al., 2000), ICV administration to
choroid plexus epithelial cells and endothelial brain microvessels (Lehtolainen et al., 2002), corpus callosum (Kukkonen et al., 2003), choroid plexus cells (Laitinen et al., 2005a) and with neuronal promoter, astrocytes (Wang and Wang, 2006). Biodistribution studies after ICV administration showed RT-PCR positivity in the spleen, heart and lungs indicating escape from the cranium, with the conclusion of baculovirus escape through the arachnoid villi (Lehtolainen et al., 2002). There has also been report on baculoviral traffic via axons of neurons (Li et al., 2004), thus contributing to viral biodistribution in a previously unexpected manner.
In our study, after ICV injection of Baavi the remaining radioactivity was lower as compared to the chelate control, further supporting the theory of systemic escape of the baculovirus (Lehtolainen
th technetium. In comparison to the MRI, the poor anatomical resolution of
5.3.4 Other administration routes and conclusions
et cellular receptors, a hypothesis of binding with charge
to the presented administration methods described, some other routes have also been used wi
sical complement pathway and degradin
ainen et al., 2002) and av
et al., 2002). We could detect a signal in the kidneys 65 minutes after the injection (III/
fig 3), indicating escape of the virus from the CSF circulation, possibly by the arachnoid villi and the lymphatic system (Kida et al., 1993). However, due to the small injected volume and consequent low radioactivity (5-8 MBq), the limit for direct detection of lymphatic circulation or neuronal transport was too high wi
SPECT prevents further pinpointing of the viral biodistribution in the brain. However, the use of another high-energy isotope such as 111Indium might provide more accurate results in the future.
While the specific interactions between baculovirus surface proteins and targ leading to baculovirus tropism, are not exactly known, there is
related manner via heparan sulphate residues instead of specific receptor (van Loo et al., 2001), supported by a large number of susceptible cell-lines in vitro (Song et al., 2003). If true, baculoviral transduction pattern should be wide also in vivo, if not hindered by other factors. Previously it has been reported that baculovirus transduction in vivo is inhibited by the blood complement system (Hofmann and Strauss, 1998), forcing researchers to discover elaborate methods to circumvent this problem. In addition
th baculoviruses.
Rabbit carotid arteries have been transduced with baculovirus by using silicon collars as artificial virus reservoirs (Airenne et al., 2000), a method enabling the protection of the baculovirus. Subretinal and intravitreal injections of baculoviruses to mouse eye demonstrated that transduction in immuno-privileged areas other than the brain are possible (Haeseleer et al., 2001).
Similarly, the immunopriviledged testis (Ferguson and Griffith, 1997) has been shown to be accessible to baculovirus transduction (Tani et al., 2003b). In a vaccination study, intranasal and subcutaneous administration were utilized to produce immunoresponse towards displayed antigens (Facciabene et al., 2004). Finally, lumbar puncture, routinely used for spinal anaesthesia, has been shown to be an effective method to reach dorsal root ganglion neurons and induce neuronal regeneration of peripheral nerves (Tani et al., 2003a).
To conclude, as compared to the IV injection the IP injection might result in gene delivery to the organs in the abdomen without severely activating the clas
g the virus. Recently, it has been shown clinically that an IP administration in the treatment of ovarian cancer can be used to reduce the side effects while reaching the therapeutic window with a smaller dose as compared to IV administration (Armstrong et al., 2006). The results of this study suggest that IP injection of baculoviral might offer a new method to target kidney and treat nephronal diseases (Osada et al., 1999).
Additionally the data from this study indicates that the baculovirus is able to spread systemically via the lymphatics, as suggested by earlier studies with baculovirus (Lehtol
idin/biotin liposomes (Medina et al., 2006). Yet more studies are needed to clarify the possible baculovirus traffic via the lymphatic system and the effects on gene therapy treatment and safety.
5.4 Article IV
While baculovirus has several benefits as compared to other major gene delivery vectors, one of the downfalls is the mediocre or poor overall transduction efficiency. This problem can be circumvented by concentrating the virus to target cells, using various targeting methods as discussed in chapter 2.3.1 or by including transduction enhancing elements, such as histone eacetylase inhibitors (e.g. sodium butyrate) or genetic elements increasing transduction efficiency (e.g. transcriptional regulatory elements).
Baculoviral cell entry requires endosomal maturation, after which the capsids are released and transported to the nucleus. It has been reported that half of the capsids reach the nucleus, while the rest are degraded in lysosomes (Dee and Shuler, 1997). Therefore improvements in this step are likely to also improve the transduction efficiency. In this study we analyzed the effects of displaying a truncated 21 aa version of VSV-G together with native gp64 to baculoviral transduction.
5.4.1 VSV-GED displaying virus
VSV-G pseudotyping has been used to widen the retroviral tropism, however the VSV-G has recently been shown to offer increased resistance towards complement inactivation of baculoviruses (Tani et al., 2003b) and restore the productive infection, replication and propagation of gp64-null baculoviruses (Mangor et al., 2001). The VSV-G membrane proximal stem region (IV/ Figure 1), in addition to the transmembrane and cytoplasmic domains (VSV-GED) has been utilized also as a display platform, to create a non-polarly distributed fusion protein with GFP (Chapple and Jones, 2002) and ZZ-domain on baculovirus (Ojala et al., 2004).
Since there have been reports of VSV-GED improving membrane fusion capabilities (Jeetendra et al., 2002), VSV-GED was included in the baculovirus together with native gp64 glycoprotein.
VSV-GED display was confirmed from the concentrated viruses by immunoblotting with VSV-G antibody (IV / fig 2), which recognizes the 15 carboxy-terminal aminoacids of the VSV-GED. In agreement with a previous study (Robison and Whitt, 2000), trimer of VSV-GED was also detected on the immunoblot. To examine the ratio of total viral particles vs. infective virus
VSV-GED display was confirmed from the concentrated viruses by immunoblotting with VSV-G antibody (IV / fig 2), which recognizes the 15 carboxy-terminal aminoacids of the VSV-GED. In agreement with a previous study (Robison and Whitt, 2000), trimer of VSV-GED was also detected on the immunoblot. To examine the ratio of total viral particles vs. infective virus